MUTATIONEN DES FLT3 GENES IN AKUTER MYELOIDER LEUKÄMIE · Mutationen Resistenzen gegenüber...
Transcript of MUTATIONEN DES FLT3 GENES IN AKUTER MYELOIDER LEUKÄMIE · Mutationen Resistenzen gegenüber...
MUTATIONEN DES FLT3 GENES IN AKUTER MYELOIDER LEUKÄMIE
Ksenia Bagrintseva
GSF - National Research Center for Environment and Health
Clinical Cooperative Group “Leukemia”
Department of Medicine III, University Hospital Grosshadern, Ludwig-Maximilians
University,
Direktor: Prof.Dr.W.Hiddemann
MUTATIONEN DES FLT3 GENES IN AKUTER MYELOIDER LEUKÄMIE
Dissertation
zum Erwerb des Doktorgrades der Medizin (Dr.med.)
an der Medizinischen Fakultät der
Ludwig-Maximilians-Universität zu München
Vorgelegt von
Ksenia Bagrintseva
aus
Moskau, Russland
2005
Mit Genehmigung der Medizinischen Fakultät
Der Universität München
1. Berichterstatter: Prof.Dr.W.Hiddemann
2. Berichterstatter: Prof.Dr.H.-G.Klobeck
Mitberichterstatter: Priv.Doz.Dr.G.Meinhardt
Prof.Dr.A.Borkhardt
Mitbetreuung durch den
Promovierten Mitarbeiter: Dr.K.Spiekermann
Dekan: Prof.Dr.med.Dr.h.c.K.Peter
Tag der mündlichen Prüfung: 24.02.2005
MUTATIONS OF THE FLT3 GENE IN ACUTE MYELOID LEUKEMIA
Contents 1. ABSTRACT......................................................................................................1 2. ZUSAMMENFASSUNG ...................................................................................2 3. INTRODUCTION..............................................................................................4 3.1 Acute myeloid leukemia (AML).......................................................................5
3.1.1 Classification of AML..............................................................................5
3.2 Receptors tyrosine kinase (RTK)....................................................................7
3.2.1 Signalling through RTK...........................................................................7
3.2.2 Class III RTKs: role in leukemogenesis ..................................................8
3.2.3 Mutations of FLT3 in AML ......................................................................9
3.3 RTK inhibitors.................................................................................................12
4. THE GOAL OF STUDY....................................................................................14
5. MATERIALS AND METHODS.........................................................................15 5.1 Chemicals.......................................................................................................15
5.2 Kits .................................................................................................................15
5.3 Laboratory equipment ....................................................................................16
5.4 Cells ...............................................................................................................17
5.5 Materials used for the cell culture...................................................................17
5.5.1 Cell culture media...................................................................................17
5.5.2 Culture flasks .........................................................................................18
5.6 Antibodies for Western Blot ............................................................................18
5.7 Software .........................................................................................................19 5.8 Cell culture methods.......................................................................................19
5.8.1 Cell culture techniques ...........................................................................19
5.8.2 Cell quantification and evaluation of viability ..........................................19
5.8.3 Storage of cells.......................................................................................20
5.8.4 Proliferation assay..................................................................................20
5.8.5 Transfection of the 293 cells...................................................................21
5.8.6 Stable transduction of Ba/F3 cells ..........................................................21
5.8.7 Subculturing of adherent cells ................................................................21
5.8.8 Cell sorting .............................................................................................21
5.8.9 FACS analysis........................................................................................22
5.8.7.1 Expression of CD135 by flow cytometry ..............................................................22
5.8.7.2 Apoptosis analysis ..............................................................................................22
5.8.10 Development of SU5614 resistant cell lines .........................................22
5.9 Biochemical methods .....................................................................................22
5.9.1 Buffers and solutions ..............................................................................22
5.9.2 Cell lysis .................................................................................................24
5.9.3 SDS polyacrylamidgelelectrophoresis-PAGE .........................................24
5.9.4 Western Blot analysis .............................................................................25
5.9.5 Protein estimation...................................................................................25
5.10 Immunological methods................................................................................26
5.10.1 Immunoprecipitation .............................................................................26
5.11 Molecular biology methods...........................................................................26
5.11.1 Buffers and solutions ............................................................................26
5.11.2 Polyacrylamide gel electrophoresis ......................................................27
5.11.3 Digestion of DNA..................................................................................27
5.11.4 Procedure for agarose gel electrophoresis...........................................28
5.11.5 Polymerase chain reaction ...................................................................28
5.11.6 Preparation of the competent E.coli......................................................29
5.11.7 Transformation of the competent E.coli ................................................29
5.11.8 DNA purification....................................................................................29
5.11.8.1 Purification of genomic DNA..............................................................................29
5.11.8.2 Mini-preparation .................................................................................................30
5.11.8.3 Maxi-preparation ................................................................................................30
5.11.9 RNA isolation........................................................................................30
5.11.10 Reverse transcription RNA to cDNA...................................................31
5.11.11 In vitro mutagenesis ...........................................................................31
5.12 Primer...........................................................................................................33
5.13 Plasmids.......................................................................................................34
6. RESULTS.........................................................................................................35 6.1 FLT3-TKD and FLT3-LM in patients with AML ...............................................35
6.2 A new and recurrent mutation of FLT3: 840GS..............................................36
6.2.1 Detection of the FLT3-LM in exon 20 .....................................................38
6.2.2 Generation of FLT3-840GS Ba/F3 cell lines...........................................39
6.2.3 Expression and hyperphosphorylation of FLT3-840GS transformed
Ba/F3 cells................................................................................................... 40
6.2.4 Transforming potential............................................................................40
6.2.5 FLT3-840GS activates the MAPK-STAT signalling pathway of FLT3.....41
6.2.6 Sensitivity of the new mutation FLT3-840GS to the selective PTK.........41
inhibitor SU5614.....................................................................................42
6.3 A new point mutation in the juxtamembrane region of the FLT3: FLT3 -
V592A............................................................................................................43
6.3.1 MM1 and MM6 cell lines.........................................................................43
6.3.2 Generation of the Ba/F3 cell lines ..........................................................43
6.3.3 Sensitivity of the new mutation FLT3-V592A to the selective PTK
inhibitor SU5614....................................................................................45
6.3.4 Importance of the Y589/591 in FLT3-V592A signalling ..........................45
6.4 Sensitivity of different FLT3-TKD point mutations to the PTK inhibitor
SU5614 ........................................................................................................47
6.4.1 The FLT3-TKD mutants D835H/Q/V/Y induce IL-3-independent growth
in Ba/F3 cells.........................................................................................47
6.4.2 The FLT3-TKD mutants activate the STAT5 and MAPK signalling
pathways ................................................................................................48
6.4.3 FLT3-TKD mutants differ significantly in their sensitivity to the
growth inhibitory activity of the FLT3 PTK inhibitor SU5614 ...................49
6.4.4 SU5614 induces apoptosis in FLT3-ITD and FLT3-D835Y, but not in
FLT3- D835H transformed cells ..............................................................51
6.4.5 The FLT3 PTK inhibitor SU5614 downregulates autophosphorylation
of the FLT3-ITD, but not the FLT3-D835H receptor mutant.....................52
6.5 Mechanisms of drug resistance to SU5614....................................................54
6.5.1 Generation of SU5614-resistant Ba/F3 FLT3 ITD cells ..........................54
6.5.2 Activation of STAT5 and MAPK in FLT3-ITD-R1-4 cell lines in
response to SU5614...............................................................................56
6.5.3 Ba/F3 FLT3-ITD-R1-4 cells are resistant to the FLT3 PTK inhibitor
AG1295, but not to Genistein and Ara-C .................................................57
6.5.4 Expression of FLT3 protein in resistant cell lines ...................................58
6.5.5 The SU5614 resistant ITD-R1-4 cell lines aquired distinct FLT3-TKD
mutations .................................................................................................59
6.6 ‘’Dual’’ FLT3 ITD-TKD mutants are resistant to SU5614 ................................62
6.6.1 The FLT3 ITD-TKD dual mutation is responsible for the SU5614
resistant phenotype in FLT3-ITD-R1-4 cells ..........................................62
6.6.2 Dual mutants are resistant to SU5614 independent from the kind of
mutation in juxtamembrane region .........................................................65
6.6.3 The presence of FLT3-ITD and FLT3-TKD mutations on two alleles
do not confer SU5614 resistance ...........................................................66
7. DISCUSSION...................................................................................................68 7.1 Functional characterization of two ‘‘new’’ FLT3 mutations in AML:V592A
and 840GS .....................................................................................................68
7.2 FLT3 as a molecular target for selective therapeutic approaches in AML ......70
7.3 ‘’Dual’’ mutations in the FLT3 gene as a cause of drug resistance to FLT3
PTK inhibitors .................................................................................................72
7.4 The FLT3-ITD/TKD dual mutation is responsible for the PTK inhibitor
resistance .......................................................................................................73
7.5 Different kind of mutations in the JM region of FLT3 induce FLT3 PTK
inhibitor resistance in dual mutant receptors ..................................................74
8. ACKNOWLEDGMENTS ..................................................................................76
9. PUBLICATIONS ..............................................................................................77
10. ABBREVIATIONS..........................................................................................79 11. REFERENCES...............................................................................................80 12. CURRICULUM VITAE....................................................................................86
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1. ABSTRACT (English) Activating mutations in the juxtamembrane domain (FLT3-length mutations, FLT3-LM)
and in the protein tyrosine kinase domain (TKD) of FLT3 (FLT3-TKD) represent the
most frequent genetic alterations in acute myeloid leukemia and define a distinct
molecular entity in AML.
In this work two new mutations in FLT3 gene are described: the first is a 6-bp
insertion on the activation loop of FLT3 between codons 840 and 841 of FLT3 (FLT3-
840GS) in two unrelated patients with AML and the second is a single and identical
T→C nucleotide exchange in both the MM6 and MM1 cell lines resulting in a V592A
mutation.
In functional analyses we could show that these mutants are hyperphosphorylated on
tyrosine and confer interleukin-3 independent growth to Ba/F3 cells, which can be
inhibited by a specific FLT3 PTK inhibitor.
These results show for the first time that in addition to known mutations in the JM and
the catalytic domain, further activating mutations exist in the FLT3 gene.
Furthermore we could show that distinct activating FLT3-TKD mutations at position
D835 mediate primary resistance to FLT3 PTK inhibitors in FLT3 transformed cell
lines. In the presence of increasing concentrations of the FLT3 PTK inhibitor SU5614
inhibitor resistant Ba/F3 FLT3-internal tandem duplication (ITD) cell lines (Ba/F3
FLT3-ITD-R1-4) were generated and characterized by a 7-26 fold higher IC50 to
SU5614 compared to the parental ITD cells. The detailed molecular characterization
of ITD-R1-4 cells demonstrated that specific TKD mutations (D835N and Y842H) on
the ITD background were acquired during selection with SU5614. Introduction of
these “duale” ITD-TKD, but not single D835N or Y842H FLT3 mutants in Ba/F3 cells
restored the FLT3 -inhibitor resistant phenotype.
These data show that pre-existing or acquired mutations in the PTK domain of FLT3
can induce drug resistance to FLT3 PTK inhibitors in vitro. These findings provide a
molecular basis for the evaluation of clinical resistance to FLT3 PTK inhibitors in
patients with AML.
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2. ZUSAMMENFASSUNG
Aktivierende Mutationen in der juxtamembranösen Region (FLT3-Längenmutationen,
FLT3-LM; FLT3- Interne Tandemduplikation, FLT3-ITD) und der Tyrosinkinase-
Domäne (FLT3-TKD, FLT3-D835) von FLT3 stellen die häufigsten genetischen
Alterationen in der AML dar und definieren eine klinisch-prognostische-Subgruppe in
der AML.
In der vorliegenden Arbeit wurden zwei neue Mutationen in FLT3 identifiziert und
charakterisiert. Die erste Mutation (FLT3-840GS) wurde in zwei Patientenproben
nachgewiesen. Moleculargenetisch stellte diese eine Längenmutation in Exon 20 dar,
und war durch eine 6 bp-grosse Insertion bzw. Glycin+Serin Insertion in der
Aktivierungsschleife der katalytischen Domäne zwischen den Kodons 840 und 841
nahe der Aktivierungsschleife verursacht. Die zweite Mutation, eine aktivierende
Punktmutation in der JM-Region von FLT3 (FLT3-V592A) wurde in den AML-Zelllinien
MonoMac 6 und MonoMac 1 identifiziert. Die funktionelle Analyse ergab, dass diese
Mutanten eine konstitutive Tyrosinphosphorylierung aufweisen und zu einem IL-3
unabhängigem Wachstum in Ba/F3 Zellen führen, welches durch einen spezifischen
Proteintyrosinekinase (PTK) Inhibitor gehemmt werden konnte.
Diese Ergebnisse zeigen, dass neben den bisher beschriebenen noch weitere
aktivierende Mutationen in der JM- und der katalytischen Domäne in FLT3 Gen
existieren.
Die weiteren Untersuchungen zeigten, dass verschiedene FLT3-TKD (D835)
Mutationen eine deutlich unterschiedliche Empfindlichkeit gegenüber selektiven FLT3
PTK Inhibitoren aufwiesen. Weiter wurde gezeigt, dass durch kontinuierliche
Exposition von FLT3-ITD transformierten Leukämiezellen mit dem FLT3 PTK Inhibitor
SU5614 in vitro resistente Mutanten generiert werden können. Die molekulare und
funktionelle Charakterisierung dieser SU5614-resistenten Zelllinien (Ba/F3 FLT3-ITD-
R1-4) ergab, dass spezifische Mutationen in der TKD-Domäne ursächlich für die
Inhibitorresistenz verantwortlich waren. Die FLT3-ITD-R1-4 Zellen wiesen somit
Doppelmutationen im FLT3-Gen (LM + TKD) auf und waren durch eine 7-26-fach
höhere IC50 gegenüber dem Inhibitor gekennzeichnet. Solche Doppelmutanten von
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FLT3 wurden mittels in- vitro- Mutagenese generiert und rekapitulieren in Ba/F3
Zellen den SU5614-resistenten Phänotyp.
Diese Ergebnisse zeigen, dass prä-existierende oder erworbene FLT3-TKD
Mutationen Resistenzen gegenüber FLT3-PTK Inhibitoren in vitro induzieren können.
Diese Befunde stellen die molekulare Basis für zelluläre Resistenzen gegenüber
FLT3-PTK Inhibitoren bei Patienten mit AML dar.
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3. INTRODUCTION
Acute myeloid leukemia (AML) is the most common form of acute leukemia
occurring in adults, comprising approximately 80 to 85% of cases of acute
leukemia diagnosed in individuals greater than 20 years of age. Currently, more
than 80% of young adults and 60% of all patients can achieve complete
remissions.
Due to the identification of leukemic fusion genes encoded by balanced
chromosomal translocations, the pathophysiology of certain AML subtypes has
been identified. Prognostic factors and disease heterogeneity can be defined using
molecular markers and provide the potential for individualizing of antileukemic
therapy in patients with AML.
3.1 Acute myeloid leukemia (AML) The pathophisiology of AML can be explained by acquired genetic changes in
bone marrow stem cells that cause a complete or partial block in normal
hematopoietic stem cell maturation. The genetic changes may involve mutations
that lead to activation of growth-promoting proto-oncogenes, inactivation of tumor
suppressor genes, or alterations of transcription factors.
Although the acquired genetic lesions that lead to leukemia are being rapidly
defined, the causative agent or environmental factor can be identified for a small
fraction of patients with AML. Nonetheless, leukemias clearly occur with increased
frequency after military or therapeutic radiation exposure, after certain types of
chemotherapy and with heavy and continuous occupational exposure to benzene.
There have been a number of large, survey-type epidemiologic studies that have
attempted to link a variety of environmental exposures to leukaemia incidence.
But, as in many epidemiologic studies, it is difficult to quantify the degree of
exposure to various environmental insults. There may be a small increased risk
associated with cigarette smoking, whereas exposure to electromagnetic radiation
seems an unlikely cause of AML. However, occupational exposures, particularly to
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benzene or petrochemicals have been implicated in the development of AML.
Alkylating agents used in Hodgkin’s disease, multiple myeloma, ovarian cancer,
and colon cancer have been associated with loss of chromosome 5 and 7,
whereas tenoposide use in childhood acute lymphoblastic leukaemia (ALL) and
high-dose anthracycline/cyclophosphamide combinations have caused alterations
of chromosome 11q23. With the few exceptions noted above, however, there is no
clear relationship between environmental exposures and the occurrence of acute
leukemia.
3.1.1 Classification of AML
In 1976, a group of morphologists from France, the United States, and Great
Britain (FAB) suggested a classification system designed to define distinct
subtypes of AML and ALL (M0-M7) (Table 1).
Table 1: FAB classification of AML. Tdt-terminal deoxinucleotide transferase, HLA-DR-human leukocyte antigen D-related (from ‘’Cancer Medicine’’)
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The diagnosis of AML requires that myeloblasts constitute 20% (based on a recent
World Health Organization classification system) or more of bone marrow cells or
circulating white blood cells. Neoplastic promyelocytes, monoblasts, or
promonocytes and megakaryoblasts are included in this calculation and their
presence defines the various subtypes of AML. Monoclonal antibodies directed
against antigen groups termed cluster designation (CD) considered to be restricted
to cells committed to myeloid differentiation are also helpful. Antibodies against
CD11b, CD13, CD14 and CD33 are used most commonly.
The new WHO classification retains the morphologic subgroups of the FAB
system, but has created new categories which recognize the importance of certain
cytogenetic translocations as predictors of response to therapy (Table 2).
Table 2: WHO classification of AML (from ‘‘Cancer Medicine’’)
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3.2 Receptor tyrosine kinases 3.2.1 Signaling through receptor tyrosine kinases
The extracellular signal proteins that act through receptor tyrosine kinases consist
of a large variety of secreted growth factors and hormones. The human genome
as currently sequenced, is thought to contain 90 tyrosine kinase genes, of which
58 are of the receptor type. Receptor tyrosine kinases can be classified into more
than 16 structural families, each dedicated to its complementary family of protein
ligands.
In all cases, the binding of the signal protein to the ligand-binding domain on the
outside of the cell activates the intracellular tyrosine kinase domain (Figure 1).
Once activated, the kinase domain transfers a phosphate group from ATP to
selected tyrosine side chains, both on the receptor proteins themselves and on
intracellular signaling proteins that subsequently bind to the phosphorylated
receptor. The tyrosine phosphorylated receptor serves as a docking site for an
array of intracellular signalling molecules, including the GTPase-activating protein
(GAP), the p85 subunit of phosphatidyl-inositol 3’-kinase (PI3K), phospholipase C-
the protein tyrosine phosphatase SHP1, Grb2 and Src-like non-receptor ,(ץ-PLC) ץ
kinases; (Rosnet, Buhring et al. 1996)Porter and Vaillancourt, 1998). These
activated proteins then initiate serine/threonine phosphorylation cascades resulting
in activation of transcription factors that determine a variety of cell responses,
including cell maintenance, mitogenesis, migration and differentiation (Claesson-
Welsh, 1994). All RTKs contain an evolutionary conserved kinase domain, and the
available crystal structures of RTK kinase domain exhibit a conserved structure
(Hubbard 1994, Mohammadi 1996, McTigue 1999). Crystallografic studies of the
insulin receptor (IR), solved in both active and inactive conformations, reveal a
twofold mechanism of activation (Hubbard 1994, Hubbard 1997). In the inactive
conformation, the activation loop of IR occupies the active site, blocking substrate
access and/or ATP binding. Upon phosphorylation of tyrosine residues within the
activation loop, structural changes occur that reposition the activation loop away
from the active site, resulting in an active conformation. Mutations both in the
activation loop and in other regions of the kinase domain have been identified in
several RTKs, which most probably stabilize an active kinase conformation.
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Figure 1 Protein tyrosine kinase activation mechanisms. Left: inactive conformation of RPTK. Right: ligand-induced receptor dimerization and tyrosine autophosphorylation (From P.Blume-Jensen and T.Hunter, Nature 411:355-365, 2001).
3.2.2 Class III receptor tyrosine kinases: role in leukemogenesis
The class III RTKs, which include FMS, KIT, FLT3, PDGFRα and PDGFRβ play an
important role in normal hematopoiesis (with the exception of PDGFR). FMS, the
receptor for the macrophage colony-stimulating factor (M-CSF), is crucial for the
growth and differentiation of the monocyte-macrophage-osteoclast lineage (Sherr,
1990). FLT3 and KIT are both required for the survival, proliferation and
differentiation of hematopoietic progenitor cells, while c-kit is also important for the
growth of mast cells, melanocytes, primordial germ cells and interstitial cells of
Cajal. (Drexler 1996; Lyman and Jacobsen 1998)The hematopoietic functions of
PDGFRβ are less well defined, although the receptor and its ligand probably play
a significant role in megekaryocytopoiesis (Yang, 1997).
The class III RTKs have recently been linked to the pathogenesis of an increasing
number of hematological malignancies. KIT mutations, for example, have been
shown to be causative in adult-type mastocytosis, as well as being associated with
acute myeloid leukaemia and sinonasal lymphomas. Indeed, Longley et al
(Longley, Reguera et al. 2001)have proposed a classification of mast cell disease
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based on the nature of the c-kit mutations. FLT3 is a gene most commonly
mutated in AML and the presence of FLT3-ITD appears to be a second strongest
independent prognostic factor in the disease after cytogenetic. (Kiyoi, Naoe et al.
1999) The role of PDGFRβ-fusion genes in bcr-abl-negative chronic
myeloproliferative disorders is still unfolding, but at least eight partner genes have
been identified.
3.2.3 Mutations of FLT3 in AML Recent advances in genetics have shown that not only chromosome abnormalities
but also molecular alterations are useful to characterize and subclassify acute
myeloid leukemia (AML). For example, partial tandem duplication within the MLL
gene (MLL-PTD) has been shown to define a subgroup of AML patients with
unfavourable clinical outcome. (Schnittger, Kinkelin et al. 2000)
FLT3 (fms-like tyrosine kinase-3; STK1, human stem cell tyrosine kinase-1 or FLK-
2, fetal liver kinase-2) is a receptor tyrosine kinase (RTK), which is known to play
an important role in normal hematopoiesis and leukemogenesis. (Matthews,
Jordan et al. 1991; Rosnet, Marchetto et al. 1991)FLT3 has strong sequence
similarities to other members of the class III RTK receptor family. This protein
family include FLT3, FMS, platelet-derived growth factor receptor (PDGFR), and
KIT and characterized by an extracellular domain comprised of 5
immunoglobulinelike (IG-like) domains and by a cytoplasmic domain with a split
tyrosine kinase motif (Figure 2).
Figure 2: FLT3 receptor.
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IgD- immunoglobuline domain; JM- juxtamembrane domain; KI-kinase insert; TK1, TK2-tyrosine kinase 1and 2 domains; LM-length mutation; MT-mutation
FLT3 is expressed in a variety of human and murine cell lines of both myeloid and
B-lymphoid lineage. In normal bone marrow, expression appears to be restricted
to early progenitors, including CD34+ cells with high levels of expression of CD117
(C-KIT). FLT3 is also expressed at high levels in a spectrum of hematologic
malignancies including 70% to 100% of acute myelogenous leukemia (AML) of all
FAB subtypes, B-precursor cell acute lymphoblastic leukemia (ALL), a fraction of
T-cell ALL, and chronic myelogenous leukemia (CML) in lymphoid blast crisis.
These data indicate that FLT3 expression may play a role in the survival or
proliferation of leukemic blasts. (Carow, Levenstein et al. 1996; Drexler 1996)
Both FLT3 overexpression and activating mutations in the FLT3 gene can be
found in patients with AML. 20-25% of patients with AML carry activating FLT3-LM
in the juxtamembrane domain. (Nakao, Yokota et al. 1996; Kottaridis, Gale et al.
2001; Reilly 2002; Schnittger, Schoch et al. 2002)In another 7% mutations at
codons 835/836 in the activation loop of FLT3 can be detected (FLT3 tyrosine
kinase mutations, FLT3-TKD).; (Abu-Duhier, Goodeve et al. 2001; Thiede, Steudel
et al. 2001; Yamamoto, Kiyoi et al. 2001; Thiede, Steudel et al. 2002)FLT3-LM
have been detected in all FAB subtypes of AML, with the highest reported
frequency in the M3 subtype, and less frequently in the M2 subtype. FLT3-LM are
associated with leukocytosis and poor prognosis in most, (Kiyoi, Naoe et al. 1999;
Xu, Taki et al. 1999; Rombouts, Blokland et al. 2000; Thiede, Steudel et al. 2001;
Whitman, Archer et al. 2001; Yamamoto, Kiyoi et al. 2001)but not all studies.
(Schnittger, Schoch et al. 2002; Thiede, Steudel et al. 2002)
Recent data indicate that FLT3-LM are not present in systemic mast cell disease
nor in a spectrum of solid tumors. FLT3-LM have not been detected in normal
hematopoietic cells, including cord blood and bone marrow cells in which there are
high levels of expression of FLT3. In addition to length mutations in one allele of
FLT3 several studies have demonstrated biallelic mutations in FLT3, as well as
patients in whom the residual wild-type allele is lost. (Kottaridis, Gale et al. 2001;
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Whitman, Archer et al. 2001; Schnittger, Schoch et al. 2002; Thiede, Steudel et al.
2002)
FLT3-LM and FLT3-TKD mutants are constitutively autophosphorylated on
tyrosine residues, causing activation of signal transducer and activator of
transcription (STAT)-5 and mitogen-activated protein (MAP) kinases. (Hayakawa,
Towatari et al. 2000; Mizuki, Fenski et al. 2000; Tse, Mukherjee et al. 2000; Tse,
Novelli et al. 2001; Spiekermann, Dirschinger et al. 2003)In addition, transduction
of FLT3-ITD and TKD mutants in murine IL-3-dependent cell lines, such as Ba/F3
and 32D induces IL-3 independent growth (Fenski, Flesch et al. 2000; Hayakawa,
Towatari et al. 2000; Mizuki, Fenski et al. 2000; Tse, Mukherjee et al. 2000;
Yamamoto, Kiyoi et al. 2001)in vitro . Injection of 32D or Ba/F3 cells stably
transfected with constitutively activated FLT3 into syngeneic recipient mice results
in the development of a leukemic phenotype. (Mizuki, Fenski et al. 2000)
Furthermore, retroviral transduction of FLT3-ITD constructs in primary murine
bone marrow cells induces a myeloproliferative phenotype in a mouse bone
marrow transplantant model. (Kelly, Liu et al. 2002)These data demonstrate that,
although FLT3-ITDs have been associated primarily with AML in humans, FLT3-
ITD alone are not sufficient to induce AML in primary hematopoietic cells.
Furthermore, a kinase-dead mutant of FLT3 in the context of FLT3-ITD abrogates
the myeloproliferative disease, indicating an absolute requirement of FLT3 kinase
activity for the myeloproliferative phenotype in this model. No difference in biologic
activity of FLT3-ITD mutants have been found in cell culture or murine models,
despite considerable variation in repeat length that ranges from several to more
than 50 amino acids.
Thus, both LM and TKD mutations in the FLT3 gene result in constitutive
activation of the kinase activity and downstream targets including STAT5,
RAS/MAPK, and PI3K/AKT. Expression of FLT3 activating mutations in primary
hematopoietic cells results in a myeloproliferative phenotype, and indicates, that
additional mutations are required for development of AML.
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3.3 RTK inhibitors
Although activating FLT3 mutations seem not be sufficient to cause an AML
phenotype they represent a potential therapeutical target. Targeted inhibition of
aberrant kinase signalling can be an effective therapeutic intervention in
hematologic malignancies, as evidenced by hematologic and cytogenetic
responses in chronic myelogenous leukemia (CML) and CML blast crisis patients
treated with the BCR-ABL kinase inhibitor imatinib mesylat (STI571, Gleevec®).
(Druker, Sawyers et al. 2001; Druker, Talpaz et al. 2001; Savage and Antman
2002)An analogous kinase inhibitor strategy might have therapeutical potential in
AML patients with activating mutations in the FLT3 gene.
In the past decade, many laboratories embarked on projects aimed at generating
compounds that specifically inhibit the activity of the signalling cascades triggered
by tyrosine kinases. Compounds with selective activity to class III RTK in vitro
include AG1295, (Levis, Tse et al. 2001)SU5416, (Fong, Shawver et al. 1999;
Spiekermann, Faber et al. 2002)SU5614 (Yee, O'Farrell et al. 2002; Spiekermann,
Dirschinger et al. 2003)and CT53518. (Kelly, Yu et al. 2002)Three compounds
(CEP-701, SU11248 and PKC412) with in vivo activity are currently evaluated in
phase I/II clinical trials in patients with AML and have shown promising results.
(Foran, O'Farrell et al. 2002; Foran, Paquette et al. 2002; Smith, Levis et al. 2002;
Stone, Klimek et al. 2002)
The PTK inhibitor imatinib mesylate has high clinical activity in BCR-ABL-positive
CML in chronic phase and blast crisis as well as in BCR-ABL-positive acute
lymphoblastic leukemia and gastrointestinal stromal tumors (GISTs) carrying
activating KIT mutations. (Druker, Talpaz et al. 2001; van Oosterom, Judson et al.
2001; Kantarjian, Sawyers et al. 2002)However, small molecule protein tyrosine
kinase inhibitors may lead to drug resistance. (Sawyers 2001)Recent studies have
indicated that resistance to imatinib may be multifactorial. (Mahon, Deininger et al.
2000; Weisberg and Griffin 2000)In certain BCR-ABL-positive cell lines, resistance
was associated with amplification of the fusion gene. (le Coutre, Tassi et al. 2000;
Mahon, Deininger et al. 2000; Weisberg and Griffin 2000)Additionally,
overexpression of the alpha 1 acid glycoprotein was detected in some resistant
- 13 -
cells. (Gambacorti-Passerini, Barni et al. 2000)At least ten different mutations in
the ABL gene that induce imatinib resistance in vitro were found in patients with
BCR-ABL-positive CML and ALL. (Gorre, Mohammed et al. 2001; Barthe, Gharbi
et al. 2002; Branford, Rudzki et al. 2002; Hofmann, Jones et al. 2002;
Roumiantsev, Shah et al. 2002; Shah, Nicoll et al. 2002; von Bubnoff, Schneller et
al. 2002)Although FLT3 PTK-inhibitors have shown promising results in phase I/II
clinical studies in patients with AML, it is unknown whether similar mechanisms of
resistance exist for FLT3 PTK inhibitors.
Previously, our group and others have demonstrated that the small molecule PTK
inhibitor SU5614 is a selective inhibitor of FLT3 that induces growth arrest and
apoptosis in FLT3 transformed cells and AML-derived cell lines expressing a
constitutively activated FLT3. (Yee, O'Farrell et al. 2002; Spiekermann,
Dirschinger et al. 2003)
- 14 -
4. THE GOAL OF STUDY
FLT3 is known to be the most mutated gene in AML. FLT3-LM were found in 20-
25% of patients with AML and 7% of patients carry FLT3-TKD mutations. The high
incidence of FLT3 mutations in AML makes it an attractive target for therapeutic
interventions. The important questions here are, whether other FLT3 mutations
can also be found in patients with AML and whether the kind of mutation in FLT3
might be important for the sensitivity to PTK inhibitors.
It was recently shown that conformational changes in the activation loop of KIT by
the D816V mutation led to resistance of this mutant to PTK inhibitor imatinib.
Therefore, we wanted to determine, whether FLT3-ITD and different point
mutations in TKD of FLT3 may differ in their sensitivity to FLT3 PTK inhibitors.
The development of drug resistance is major problem of chemotherapy in patients
with AML. Previous studies on the BCR-ABL PTK inhibitor imatinib have shown
that drug resistance may be multifactorial and include: overexpression of the
fusion gene, development of additional mutations and overexpression of the alpha
1 acid glycoprotein. Although FLT3 PTK inhibitors have shown promising results in
phase I/II clinical studies in patients with AML, the similar mechanisms of drug
resistance might occur. In this study Ba/F3 FLT3-ITD transformed cells were used
as a model system and were incubated in the presence of the increasing
concentration of the FLT3 PTK inhibitor. Furthermore, these cells were used to
characterize potential mechanisms of drug resistance in vitro.
It was the aim of this study to detect and characterize new mutations in the FLT3
gene and answer the questions whether FLT3-TKD mutations can confer primary
resistance to PTK inhibitors and whether the secondary resistance to these
inhibitors can develop in FLT3-ITD transformed hematopoietic cell lines.
- 15 -
5. MATERIALS AND METHODS
5.1. Chemicals * Corrosive; ** Toxic; *** Comburant
Acetic acid (Merck, Darmstadt, Germany) *
Acrylamide/Bisacrylamide 40% (Roth, Karlsruhe, Germany) **
Agarose (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Ammonium peroxidisulfate (APS) (Bio-rad, Munich, Germany)
Aprotinin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Bovine serumalbumin (BSA) (Fluka, Buch, CH)
Ethanol (Merck, Darmstadt, Germany) ***
Glycerin (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
HEPES (N-(2-Hydroxyethyl)piperazine-N´-(2-ethanesulfonic acid)) (Sigma-Aldrich
Chemie GmbH, Taufkirchen, Germany)
Leupeptin A (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
2-mercaptoethanol (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) **
Methanol (Merck, Darmstadt, Germany) ***
Pepstatin A (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich Chemie GmbH, Taufkirchen,
Germany) **
Ponceaus (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Protein G-sepharose (monoclonal) (Amersham, Schweden)
Protein A-sepharose (polyclonal) (Upstate, USA)
Sodium Vanadate (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Sodiumdodecylsulfat (SDS) (Bio-rad, Munich, Germany)
Tetramethylethylendiamine (TEMED) (Serva, Heidelberg, Germany)
Tris(hydroxymethyl)aminomethane (Tris) (Merck, Darmstadt, Germany)
Trypan blue (Serva, Heidelberg, Germany) **
Tween 20 (Merk, Darmstadt, Germany)
5.2 Kits Annexin V/Propidium Iodide (PI) kit (Alexis, Lausen, Switzerland)
Bio-rad Protein estimation kit (Bio-rad, Munich, Germany)
- 16 -
Detection system ECL® (enhanced chemiluminescence) (Amersham Pharmacia,
Freiburg, Germany)
Qiagen Plasmid Maxi Kit (Qiagen, Germany)
RNeasy Mini Kit (Qiagen, Germany)
QIAprep-spin Miniprep Kit (Qiagen, Germany)
OmniscriptTM Reverse Transcriptase Kit (Qiagen, Germany)
5.3 Laboratory equipment Blotting chamber (Bio-rad, Munich, Germany)
Cell culture CO2 incubator (Haereus, Rodenbach, Germany)
Cell culture hood (Bio Flow Technik, Meckenhein, Germany)
Centrifuges ROTIXA/P (Hettich, Tuttlingen, Germany)
Developing machine M35X-OMAT Processor (Kodak AG, Stuttgart, Germany)
Eppendorf ultracentrifuge 2K15 (Sigma-Aldrich Chemie GmbH, Taufkirchen,
Germany)
FACSscan (Beckton Dickinson, Mountain View, CA)
Fridge (4°C, -20°C) (Siemens AG, Germany)
Fridge (-80°C) UF80-450S (Colora Messtechnik GmBH, Lorch, Germany)
Gel electrophoresis systems (Bio-rad, Munich, Germany)
Heating block BT 130-2 (HLC, Haep Labor Consult, Bovenenden, Germany)
Liquid nitrogen tank (Cryoson, Schöllkrippen, Germany)
Microscope (Carl Zeiss Jena, Germany)
Nitrocellulose membranes (Sartorius, Goettingen, Germany)
pH-meter 766 (VWR International, Ismaning, Germany)
Rotor Ti 75 (Beckman, Palo Alto, CA)
Shaker (Edmund Bühler, Tübingen, Germany)
Spectophotometer Smartspec TM 3000 (Bio-rad, Munich, Germany)
Ultracentrifuge L7-65 (Beckman, Palo Alto, CA)
Vortex (Cenco, Breda, the Netherlands)
Water bath (HAAKE, Karlsruhe, Germany)
- 17 -
5.4 Cells Suspension cells
Cell type Description Origin
Ba/F3 Mouse pro B cells, IL-3 dependent murine pro B cell line, established from peripheral blood BALB/c mouse
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany)
MM1 and MM6 Human acute monocytic leukemia, established from peripheral blood
DSMZ
Adherent cells
Cell type Description Origin
293 Human embryonic kidney, adherent fibroblastoid cells
DSMZ
5.5 Materials used for the cell culture
5.5.1 Cell culture media and other reagents for cell culture
RPMI 1640 (Life Technologies Invitrogen, GIBCO BRL, Kalsruhe, Germany )
10% FCS (Fetal calf serum) (Biochrom AG seromed, Berlin, Germany)
DMSO (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Penicillin/streptomycin (GIBCO BRL, Kalsruhe, Germany)
L-glutamine (GIBCO BRL, Kalsruhe, Germany)
Phosphate Buffer Saline (PBS) (GIBCO BRL, Kalsruhe, Germany)
Trypan Blue (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)
Trypsin-EDTA (Life Technologies Invitrogen, GIBCO BRL, Kalsruhe, Germany)
5.5.2 Culture vessels and plastic ware
- 18 -
Culture flasks and plates
Container Surface area (cm 2)
Medium required (ml)
Cell volume of inoculum
Company
96-well flat plate 0,3 0,1 < 1x104 Nunc, Wiesbaden,
Germany
96-well round
plate
0,3 0,1 < 1x104 Nunc, Wiesbaden,
Germany
50 ml flask 25 5 2 x105 Greiner
Labortechnik,
Frickenhausen,
Germany
250 ml flask 75 15 6 x105 Greiner
Labortechnik,
Frickenhausen,
Germany
600 ml flask 125 30 2 x106 Greiner
Labortechnik,
Frickenhausen,
Germany
Other plastic material Centrifuge vials (15-50 ml) (Sarstedt, Nümbrecht, Germany)
Eppendorf cups (0,5-1 ml) (Eppendorf, Hamburg, Germany)
Freezing tubes (Nunc, Wiesbaden, Germany)
Micropipettes, Pipettes (Gilson, Langenfeld, Germany)
Parafilm M (American National Can, Greenwich, USA)
5.6 Antibodies for Western Blot Anti-rabbit-HRPO (horse radish peroxidase) IgG sc-2025 (Santa Cruz
Biotechnology, Santa Cruz, California, USA)
Anti-mouse-HRPO IgG sc-2027
FLT3/Flk-2 (S-18) rabbit polyclonal sc-480
- 19 -
β-Aktin mouse monoclonal
Phospho-STAT5 (Tyr 694) rabbit polyclonal (Cell Signalling Technologies, New
England Biolabs GmbH, Germany, Frankfut am Main)
STAT5 rabbit polyclonal
Phospho-p44/45 (Thr 202/Tyr 204) rabbit polyclonal
P44/45 MAPK (ERK1/2) kinase antibody rabbit polyclonal
5.7 Software Cellquest software (Bekton Dickison, Mountain View, CA)
Microsoft Word, Excel, Powerpoint, Photoshop
Winmdi 3.2
BioEdit
Adobe Illustrator
Adobe Photoshop
Tina 2.09
5.8 Cell culture methods
5.8.1 Cell culture techniques
The mammalians cells grow attached to a substratum, like for example plastic or in
suspension in plastic flasks. The plastic ware is obtained in sterile wraps from
commercial suppliers and is specially prepared for use in cell culture (see Table in
materials). All these tissue culture flasks have caps with filters which allow
gaseous exchange, allowing maintenance of correct pH (which is monitored by the
colour of the phenol red present in the medium) and the right percentage of CO2
(5%). The optimal atmosphere conditions are allowed by CO2 incubator, which
automatically control temperature and pCO2; it operates with a try of water on the
base in an attempt to maintain more than 98% relative humidity. Temperatures of
the incubator was set at 37°C and regurarly controlled. The use of plastic pipettes
and special medium (very low endotoxin) was necessary to work in an endotoxin
free condition.
- 20 -
5.8.2 Cell quantification and evaluation of viability An efficient way of counting cells and at same time evaluates the percentage of
viable cells is the technique of “dye exclusion”: this test is based on the concept
that viable cells do not take up some dyes, whereas dead cells are permeable to
these dyes. Trypan blue is the most commonly used dye, but have the
disadvantage of staining soluble proteins. In the cell culture however some miss-
leading situations such as recent trypsinization and freezing and thawing in
presence of dymethylsulphoxide (DMSO) may lead to membrane leakeness.
From each suspension cells an aliquot of 10 µl was harvested, mixed with 90 µl of
Tryplan Blue (ratio cells:trypan blue=1:1) and counted on a counting slide under
the microscope. The mean of at least three counts of viable cells (not stained with
tripan blue)/quadrant was considered and multiplied to the magnitude (104) and
the dilution factor.
5.8.3 Storage of cells
In order to minimize the cellular injury induced by freezing and thawing procedures
(intracellular ice crystals and osmotic effects), a cryoprotective agents such as
dimethyl sulphoxide (DMSO) or glycerol are added. A variable number of
suspension cells between 2-10 x 106 are spin down and resuspended in a 500 µl
10% DMSO solution (DMSO diluted in fetal calf serum). Freezing ampoules are
cooled up before and every step is performed on ice. While short-term
preservation of cell lines using mechanical freezers (-80°C) is possible, storage in
liquid nitrogen (-196°C) or its vapour (-120°C) is much preferred. Rapid thawing of
cell suspension is essential for optimal recovery.
5.8.4 Proliferation assay
Base culture medium for suspension cells (Ba/F3, MM1, MM6) was RPMI 1640
supplemented with 10 % FCS, 1% penicillin and streptomycin + 1% L-glutamine.
Both FCS as well as the antibiotics penicillin and streptomycin are aliquoted and
stored at – 20°C. Serum contains complement which may interfere in virus or
cytotoxity assays; FCS is therefore inactivated by heating it to 56°C for 45
minutes. Serum supplies many essential factors as adhesion promoting
components, nutrients and trace minerals, transport proteins (albumin, ferritin) and
- 21 -
other proteins. Cells were incubated at 37°C in a humidified atmosphere of 5%
CO2 and 95% humidity. Cells were seeded at a density of 4x104/ml growth medium in the presence or
absence of IL-3 and inhibitors or cytostatics as indicated. Viable cells were
counted at 24, 48 and 72 hours in a standard hemacytometer after staining with
trypan blue. The IC50 was defined as the concentration of inhibitor at which 50% of
cells were viable compared to cells grown in the absence of inhibitor.
5.8.5 Transfection of the 293 cells The cells were seeded at a density 5x105/ml. Transient transfections were then
carried out using the calcium-phosphate co-precipitation method with a total 6µg
DNA per 6-well (mix: 54 µl 250mM CaCl2, 54 µl HBS, pH=7.8, 6 µg DNA). 18
hours after transfection 3 ml fresh medium was added, the cells were allowed to
grow for another 30h and the retroviral supernatant was used for transduction of
Ba/F3 cells.
5.8.6 Stable transduction of Ba/F3 cells 2x105/ml Ba/F3 cells were seeded in 200µl of growth medium RPMI-1640 and
subsequently transduced once with 200µl of retroviral supernatant in the presence
of polybrene (8µg/ml). After 4-12 hours the 1 ml of medium was added to avoid the
toxic action of the polzbrene on the cells. The cells were sorted after 48-72 hours.
5.8.7 Subculturing of adherent cells
293 cells were maintained in Dulbecco’s modified Eagle Medium (DMEM; PAN,
Germany) with 10% FBS. Cells growing as monolayers on plastic surfaces are
held together and to the substratum by mucoproteins and sometimes by collagens;
in addition, many cell monolayers require divalent cations (Ca2+, Mg2+) for their
integrity. Thus for releasing cells from monolayers various protease solutions are
used, sometimes in association with chelating agent. Therefore Trypsin 0,5 EDTA
was used for subculturing of adherent cell line with following protocol: when cells
were seen to be confluent (in a 75 cm2 flask, after 2 day of culture) old medium
was washed out, cells were washed with PBS and incubated with 3 ml of Trypsin-
EDTA for 5-10 minutes in the incubator; prolonged exposure to trypsin should be
- 22 -
avoided as this damages the cells; therefore to inactive it, medium containing
serum, which contain a natural trypsin inhibitor, was added. Cells were than splittet
(subcultured) in a split ratio of 1:3 (from one 75 cm2 flask, three 225 cm2 flasks).
5.8.8 Cell sorting The FACS-Ventage system equipped with a Turbo-Sort device (Becton Dickinson,
San Jose, CA) was used to highly purify EGFP/EYFP-positive pool cells 48 hours
after infection. For this purpose, the cells were washed with PBS and then diluted
in medium containing 1µg/ml propidium jodid.
5.8.9 FACS analysis 5.8.9.1 Expression of CD135 by flow cytometry
Ba/F3 cells were incubated for 30 minutes on ice with a mouse phycoerythrin (PE)
labeled isotype-matched control antibody (Becton Dickinson) or CD135-PE
(Becton Dickinson) antibody. Viable cells were analyzed using a FacsCalibur flow
cytometer (Beckton Dickinson, San Jose, CA).
5.8.9.2 Apoptosis analysis
Assessment of apoptotic cells was carried out by annexin V/7-amino-actinomycin
D (7-AAD) staining as recommended by the manufacturer (annexin V-
phycoerythrin (PE) apoptosis detection kit, Becton Dickinson, Heidelberg,
Germany) using a FacsCalibur flow cytometer (Beckton Dickinson, San Jose, CA).
5.8.10 Development of SU5614 resistant Ba/F3-FLT3-ITD cell lines
Ba/F3 cells carrying the FLT3-ITD (W51) mutation were cultured in the presence
of increasing concentrations of SU5614 (0.2 µM to 0.8µM) over a period of at least
three months. Cells were initially cultured in the presence of 0.2 µM SU5614, with
culture medium changes every 3-4 days, for a total 27 days. Cells were then
cultured in 0.4µM SU5614 for an additional 35 days, and finally cells were grown in
medium containing 0.8µM SU5614. Then cells were analyzed for drug resistance
and further characterized.
- 23 -
5.9 BIOCHEMICAL METHODS
5.9.3 Buffers and solutions
Lysis buffer
5 ml 1M HEPES pH 7,5 3 ml 5M NaCl 0.5 ml 200 mM EGTA 20 ml 50% glycerol 1 ml Triton X-100 0.42 g NaF 0.45gNa2P2O7x10H2O(tetra-Natriumdiphosphatdekahydrat as buffer) add 100 ml aqua dest. 50 µl Aprotinin (200x) 100 µl 0.1 M PMSF (350 mg/20 ml EtOH) 100 µl 0.1 M Orthovanadat (183 g/10ml aqua dest)
10xNET
1.5 M NaCl 0.05 M EDTA pH 8 0.5 M Tris pH 7,5 0.5% Triton X-100
1x G-NET
2.5 g gelatin 100 ml 10xNET 900 ml H2O
10xTBS
10mM Tris pH 8 150 mM NaCl
1xTBST
100 ml 10xTBS 0.2 % Tween 20 Water to 1L
- 24 -
4XHNTG
200 mM HEPES pH 7,5 600 mM NaCl 0.4% Triton X-100 40% glycerol
Stripping buffer
800 ml 3xTBS 0.2% Tween 20 H20 to 1L
Laemmli-Buffer
187,5 mM Tris 6% SDS 30% glycerine bromphenolblue
Laemmli- Dithiothreitol (DTT) buffer
0.6 g of DTT 2.5 ml of Laemmli buffer
All of chemicals were obtained from Sigma or Merck (Germany).
5.9.1 Cell lysis
Cells were incubated in lysis buffer for 30 minutes at 4°C. Whole cell lysates
containing 100 µg protein as determined by the Bradford method were denatured
by boiling for 5 min in SDS sample buffer, loaded into one lane and separated by
10% SDS-PAGE.
5.9.2 SDS-Polyacrylamidgelelectrophoresis PAGE
To separate proteins according to their molecular weight (1D electrophoresis)
protein samples (whole cell lysates) are first denaturized by boiling at 95°c for 5
minutes in a SDS buffer; 40 to 100 µg of sample are loaded into a SDS gel and let
run at 150 volts in a running buffer to separate electrophoretically. Proteins with
different molecular weight and load are run differently. A marker with well-
characterized proteins allows identifying the molecular weight of unknown
proteins. Gel must be fresh prepared; gel solution solidify and polimerize quickly
(5-10 minutes) after giving APS and Temed. Gels can be kept in a humified
ambience for 1-2 days.
- 25 -
5.9.4 Western Blot analysis After electrophoresis, proteins can be transferred to nitrocellulose membranes in a
blotting puffer for 1hour at 75 volts and probed with appropriate Ab. To see all
proteins transferred, the membrane can be stained shortly with Ponceau staining,
that is a transient red staining which bind every protein. Before antibody
incubation, the membrane has to be blocked with 0.25% gelatine in NET buffer
(1xG-NET) for 1 hour and incubated overnight with appropriate antibodies diluted
1:1000. The membrane is washed 3 times for 15 minutes in TBST and then
incubated for 1 hour at room temperature in horseradisch peroxidase (HRP)
conjugated antibody diluted in the G-Net solution. Membrane was washed 3 times
for 15 minutes. Detection of proteins/antibodies complexes was achieved by the
ECL (Enhanced Luminol Reagent) system. This western blot chemiluminescence
reagent is a non-radioactive light emitting system which detects proteins
immobilized on a membrane, from the oxidation of luminol, which results in light
emission at a wave length of 428 nm, captured by a autoradiography film.
5.9.5 Protein quantification
To estimate the protein concentration Bradford assay was used: the Bradford
assay are colorimetric assay based on the differential color change of a dye in
response to various protein concentration. The Bradford assay has the advantage
of having slight chemical interferences, of beeing sensitive and rapid (only one
reagent).
Material and Method
Chemical interference
Company, reagents Technique procedure
Bio-rad protein assay (Bradford)
slight - one bottle of dye reagent (dye, phosphoric acid and methanol) stored at 4°C
- add standard, sample, blank 0,8 ml - add 0,2 ml dye reagent concentrate - after 5 minutes measure OD =595
Biorad purchase most of the reagents; required are also a spectophotometer and
cuvettes for measurements. For the protein standard lyophilized preparations of
bovine serum albumin are rehydrated in NaOH, diluted, aliquoted and stored at –
80°C.
- 26 -
5.10 Immunological methods 5.10.1 Immunoprecipitation The cells (3x107/ml) were lysed using lysis buffer. The sepharose (protein A:
polyclonal rabbit, IgG2 or protein G: IgG1 ) was washed with prechilled 1x HNTG
and diluted same volume of 1x HNTG, mixed by vortexing and stored at 4°C. To
isolate antigen on antibody bead the following steps were performed: 30 µl
sepharose, 15 µl antibody and 300 µl extracted cells were mixed and incubated on
rotation wheel for 4 hours. Than the probes were washed to remove unbound
proteins, then 20 µl Laemmli buffer were added and the samples were boiled at
100°C heating block for 8 min.
5.11 Molecular biology methods 5.11.1 Buffers and solutions
1x TBE
100 ml 5x TBE add 500 ml aqua dest.
0.5 M EDTA
18.61g EDTA 100 ml aqua dest pH 8.0
5xKCM
5ml 3M KCl 4.5 ml 1M CaCl2 7.5 ml 1M MgCl2 13 ml H2O
LB-medium
25g Luria Broth Base add 1L H2O
LB-plates
37g Luria agar add 1L H2O
LB-medium/LB agar
Powder obtained from Gibco BRL
All of chemicals were obtained from Sigma (Germany), Gibco BRL(Germany) and
Merck (Germany). Enzymes were obtained from NEB (Germany).
- 27 -
5.11.2 Polyacrylamide gel electrophoresis Polyacrylamide gels provide somewhat better resolution as well as significantly
higher capacity.
8% polyacrylamide gel: 13.3 ml acrylamide
6.25 ml bisacrylamide
5 ml 10xTBE
25.5 ml aqua dest
250 µl APS
40 µl TEMED
The gel solution was prepared in flask, poured between the gel and left to
polymerize for 30 min. After the polymerization was complete, the comb was
removed. The lower reservoir of the electrophoresis tank was filled with 1xTBE
buffer. The gel plates were clamped to the top of the electrophoresis tank and the
upper reservoir was filled with 1xTBE. DC power supply was used to pre-run and
warm the gel for 30 min at 5 V/cm (constant voltage). After that 5xloading buffer
was added to DNA samples and molecular weight markers (to 1x final) and the
probes were loaded on the gel. The gel was run at 5V/cm, and then stained for 5-
10 min in 0.5 µg/ml ethidium bromide. The DNA was detected using UV
transilluminator.
5.11.3 Digestion of DNA with restriction enzyme 0.5-1 µl Restriction enzyme (10-20 U/ul)
2 µl 10xRestriction enzyme buffer (Reb)
0.2 µl (1mg/ml) Bovine Serum Albumin (BSA, DNase free)
to 20 µl water
The DNA was added and the mix was incubated at the appropriate temperature for
required period of time (typically 2-3 hours).
5.11.4 Procedure for agarose gel electrophoresis
The correct amount of powdered agarose (Gibco BRL, Germany) was added to a
measured quantity of electrophoresis buffer (1xTBE), than the mix was heated
slurry in a microwave oven until agarose was dissolved. After cooling of this
- 28 -
solution to 50°C, the warm solution was poured into an. After the gel was
completely set (ca. 30-40 min at room temperature) 1xTBE buffer was added to
cover the gel to a depth of ca. 1 mm. The samples were mixed with 6xloading
buffer (Promega) and loaded into the slots of the submerged gel. After running the
gel was incubated for 15 min in ethidium bromide solution [0,5 ug/ml]
(Roth,Germany) and DNA was detected using UV lamp.
5.11.5 Polymerase chain reaction (PCR) 10xPCR Buffer, dNTP mix, primer solutions, and 25 mM MgCl2 were thawed on
the ice. A master mix was prepared from 10 µl 10xPCR buffer, 10 µl 25mM MgCl2,
2µl dNTPs and primers. Appropriate volumes of the master mix were dispensed
into PCR tubes and template DNA was added. The PCR tubes were placed in the
thermal cycler and programm was started.
PCR-Programm (standard):
94°C 2min
_____________
94°C 1min
58°C 1min 35-40 cycles
72°C 1min
_____________
72°C 7min
4°C ∞
5.11.6 Preparation of the competent E.coli
Over-night culture of E.coli was prepared, than the cells were transplanted in LB
broth and grown up to OD600 = 0.3-0.4 (over night). After that the cells were
centrifuged at 4000-5000 rpm for 10 min at 4°C and the pellet was resuspended
in 15ml ice-cold TSB-medium. 100µl aliquots of cells were transferred in
Eppendorf-tubes and frozen down immediately in a dry-ice for 1 hour. The aliquots
were stored at -80°C.
- 29 -
5.11.7 Transformation of competent E.coli The cells were thawed on the ice and 25µl of the cells were transferred in a tube.
After that 1-10 µl DNA (0.1-1 µl) were added and also 5µl 5xKCM and water to 50
µl. The mix was incubated on the ice for 20min and than for 10 min at room
temperature. After addition of 250 µl of LB medium, the tubes were incubated at
37C for one hour. After incubation 150 µl of the cells were spread on LB plate with
appropriate antibiotics and the plates were incubated at 37° overnight.
5.11.8 DNA purification
5.11.8.1 Purification of genomic DNA
(DNeasy protocol for cultured animal cells, QIAGEN)
The appropriate number of cells (max. 5x106) was centrifuged for 5 min at 300xg.
The pellet was resuspended in 200 µl PBS. 20 µl proteinase K and 200 µl Buffer
AL were added to the sample, mixed by vortexing and incubated at 70°C for 10
min. 200 µl ethanol (96-100%) were added to the sample. The mixture was placed
into the DNeasy spin column placed in a 2 ml collection tube and centrifuged at
6000xg for 1 min. The DNeasy spin column was placed in a 2 ml collection tube,
500 µl Buffer AW1 were added and centrifuged at 6000xg for 1 min. Than 500 µl
Buffer AW2 were added and centrifuged for 3 min at a full speed to dry the
DNeasy membrane. After that the DNeasy spin column was placed in a clean 1,5
ml or 2 ml microcentrifuge tube, 200 µl Buffer AE (elution buffer) were added
directly onto the DNeasy membrane. After incubation at RT for 1 min the DNA was
eluted.
5.11.8.2 Mini-preparation
(QIAprep Spin Miniprep Kit Protocol)
This protocol was designed for purification of up to 20µl of high-copy plasmid DNA
from 5 ml overnight cultures of E.coli in LB (Luria-Bretani) medium.
Pelleted bacterial cells were resuspend in 250µl of Buffer P1 and transferred to a
microfuge tube. 250 µl of Buffer P2 were added. 350 µl of Buffer N3 were added
and tubes were centrifuged for 10 min. The supernatants were applied to the
QIAprep column by pipetting and centrifuged for 30 sec. QIAprep spin column
were washed by adding 0.75 ml of Buffer PE and centrifuged for 30 sec and then
- 30 -
for an additional 1 min to remove residual wash buffer. Further QIAprep spin
column were placed in a clean 1.5-ml microfuge tubes. To eluate DNA, 50µl of
Buffer EB (10mM Tris-Cl, pH 8.5) were added and centrifuged for 1 min.
5.11.8.3 Maxi-preparation
(using the EndoFree Plasmid Maxi Kit from QIAGEN)
A single colony was picked from a freshly streaked selective plate and incubated
first in 2-5 ml LB medium containing the appropriate selective antibiotic for 1 hour,
then in 100 ml for additional 12-16 hours at 37C with vigorous shaking (300rpm).
After that the bacterial cells were harvested by centrifugation at 6000xg for 15 min
at 4°C and resuspended in 10 ml Buffer P1. Then10 ml Buffer P2 were added and
incubated at room temperature for 5 min. After that 10 ml chilled Buffer P3 were
added to the lysate and whole mix was poured into the barrel of the QIAfilter
Cartridge and incubated at room temperature for 10 min.
After filtration of the cell lysate into a 50 ml tube, 2.5 ml Buffer ER were added and
incubated on ice for 30 min. A QIAGEN-tip 500 were equalibrated by applying 10
ml Buffer QBT. After that the filtered lysate was placed to the QIAGEN-tip and the
QIAGEN-tip were washed with2x30 ml QC buffer. DNA elution was performed with
15 ml Buffer QN. DNA was precipitated by adding 10.5 ml room-temperature
isopropanol and centrifuged at 15,000xg for 30 min at 4C. DNA pellet was washed
with 5 ml of endotoxin-free, room-temperature 70% ethanol and centrifuged at
15,000xg for 10 min. The pellet was air-dried for 5-10 min, and redissolved in a
suitable volume of endotoxin-free Buffer TE.
5.11.9 RNA isolation, RNeasy Mini Protocol for isolation of total RNA from
animal cells. RNeasy kit (Qiagen, Hilden, Germany)
The cells (no more than 1x106) were harvested. 350 µl Buffer RLT (lysis buffer)
were added to the cells. 1 volume (350 or 600 µl) of 70% ethanol to the
homogenized lysate was added. 700 µl of each sample were placed into each
RNeasy column placed in a 2 ml collection tube and centrifuged for 15s at 8000xg.
700 µl Buffer RW1 were added to the RNeasy column and centrifuged for 15s at
8000xg. The RNeasy column was transferred into a new 2 ml collection tube. 500
µl Buffer RPE were placed onto the RNeasy column and centrifuged for 15s at
- 31 -
8000xg. After that another 500 µl Buffer RPE were placed to the RNeasy column
and centrifuged for 2 min at 8000xg to dry the RNeasy silica-gel membrane.
To elute, the RNeasy column were transferred to a new 1.5 ml collection tube, 30
µl RNase-free water was added directly onto RNeasy silica-gel membrane and
centrifuged for 1 min at 8000xg .
5.11.10 Reverse transcription RNA to cDNA
(using Dilute QIAGEN kit Omniscript Reverse Transcriptase, Hilden, Germany)
The template RNA solution was thawed on ice. The primer solutions, 10xBuffer
RT, dNTP Mix, and RNase-free water were thawed at room temperature and
than stored on ice immediately after thawing. Each solution was mixed by
vortexing, and centrifuged briefly to collect residual liquid from the sides of the
tubes. RNase inhibitor was diluted to a final concentration of 10 units/µl in ice-cold
1x Buffer RT, mixed carefully by vortexing for no more than 5 sec, and centrifuged
briefly to collect residual liquid from the sides of the tubes. Fresh master mix was
prepared (2µl 10x RT Buffer, 2 µl dNTP Mix, 2µl Oligo-dT primer, 1µl RNase
inhibitor, 1µl Omniscript Reverse transcriptase and water variable). After that
template RNA were added to the individual tubes containing the master mix and
incubated for 60min at 37°C. For analysis of shorter cDNAs by PCR or other
downstream enzymatic applications, Omniscript Reverse Transcriptase was
inactivated by heating the reaction mixture to 93°C for 5 min followed by rapid
cooling on ice.
5.11.11 In vitro mutagenesis
QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA)
Mutagenic primer design:
The primers were designed between 25 and 45 bases in length, with a melting
temperature of ≥75°C.
Tm=81.5=0.41(%GC)-675/N-%mismatch
• N is the primer length in bases
• Values for %GC and %mismatch are whole numbers
The desired mutation was closed to the middle of the primer with 10-15 bases of
template-complementary sequence on both sides.
- 32 -
Optimum primers had a minimum GC content 40% and terminated on one or more
C and G bases at the 3’-end.
Mutagenesis reaction:
10xPCR Buffer, dNTP mix, primer solutions, and 25 mM MgCl2 were thawed. A
master mix (35µl H2O, 5µl 10xPCR buffer, 3µl Quick Solution, primers) was
prepared. After that appropriate volumes were dispensed into PCR tubes. Then
template DNA and 1µl dNTPs were added to the individual tubes, containing the
master mix. After that the PCR tubes were placed in the thermal cycler and the
program was started.
Cycling parametrers (standard):
94°C 2min
80°C 1min - hot start
_____________
94°C 1min
56°C 1min 4 cycles
72°C 1min
_____________
94°C 1min
58°C 1min 14 cycles
72°C 1min
_____________
72°C 7min
4°C ∞
- 33 -
5.12 Primer Mutagenesis Primer : FLT3 V592A
5’-CAGGTGACCGGCTCCTCAGATAATGAGTACTTCTACGCTGATTTC-3’
FLT3 840GS
5’- GATATCATGAGTGATTCCGGATCCAACTATGTTGTCAGG -3’
FLT3 D835H
5’- GACTTTGGATTGGCTCGACATATCATGAGTGATTCCAACC -3’
FLT3 D835Q
5’- GACTTTGGATTGGCTCGACAGATCATGAGTGATTCCAACC -3’
FLT3 D835V
5’-GACTTTGGATTGGCTCGAGTTATCATGAGTGATTCCAACC -3’
FLT3 D835Y
5’-GACTTTGGATTGGCTCGATATATCATGAGTGATTCCAACC -3’
FLT3 D835N
5’- GACTTTGGATTGGCTCGAAATATCATGAGTGATTCCAACC -3’
FLT3 Y842H
5’-GAGTGATTCCAACCATGTTGTCAGGGGCAATG-3’
Primers for FLT3 sequencing:
pMSCV 5’- CCCTTGAACCTCCTCGTTCG-3’ 1-260bp of FLT3
cDNA
FLT3 2 5’-AAGACCTCGGGTGTGCGTTG-3’ 260-600
FLT3 3 5’-ACGCCCTGGTCTGCATATC-3’ 600-940
- 34 -
FLT3 843F 5’- CGGGCTCACCTGGGAATTAG-3’ 940-1280
FLT3 5 5’-TTTGCAATCATAAGCACCAGC- 3’ 1280-1620
FLT3 6 5’-ATACAATTCCCTTGGCACATC-3’ 1620-1960
FLT3 7 5’-AACGGAGTCTCAATCCAGG-3’ 1960-2300
FLT3 2205F 5’-CAGCATGCCTGGTTCAAGAG-3’ 2300-2640
FLT3 9 5’-AGGCATCTACACCATTAAGAG-3’ 2640-2980
5.13 Plasmids
FLT3-WT
FLT3-W51
Provided by G.Gilliland, Howard Hughes Medical School and
Institute and Bringham and Women’s Hospital Harvard
Institute of Medicine, Harvard Medical School, Boston, MA
FLT3
D835H/Q/V/Y,
FLT3-V592A,
FLT3-840GS
Prepared in K.Spiekermann group, CCG-Leukemia, GSF
National Research Center for Environment and Health,
Munich, Germany
MSCV-IRES-
EYFP/EGFP
Retroviral expression vector that contains an IRES site and
allows translation of EYFP and the construct from the same
transcript. Provided by R.K. Humphries, The Terry Fox
Laboratory, Vancouver, University of British Columbia,
Canada
- 35 -
6. RESULTS
6.1 FLT3-TKD and FLT3-LM in patients with AML Bone marrow or blood samples from 60 adult patients with newly diagnosed and
untreated AML were analysed. All were diagnosed as having AML according to
standard French-American-British (FAB) and WHO criteria and were referred to
our clinic for central cytomorphologic and cytogenetic diagnostics. The studies
abide by the rules of the local internal review board and the tenets of the revised
Helsinki protocol. Genomic DNA from the patients was screened for activating
FLT3-LM and TKD-mutations to investigate the frequency of these mutations in
AML (summarized in Table 3).
Table 3: Frequency of FLT3-TKD and FLT3-LM in patients with AML The frequency of FLT3-TKD mutations (column 3) and FLT3-LM (column 4) mutations is given in different morphologically defined AML-subgroups according to the FAB-classification. The percentage of patients carrying either the FLT3-TKD mutation or the FLT3-LM is listed in column 5.
The loss of the EcoRV restriction site at codon 835/836 by mutations (Yamamoto,
Kiyoi et al. 2001)and the previously described PCR-based assay for detection of the
FLT3-LM (Kiyoi, Naoe et al. 1999)was used for a mutation screen. Abnormal
restriction profiles at codon D835/836 were found in 6/60 (10%) of AML patients.
The presence of a TKD mutation was confirmed in all cases by nucleotide
sequencing. The mutations found at D835 were genetically heterogeneous and
resulted in substitution of D835 by glutamine (n=2), tyrosine (n=1), valine (n=1),
alanine (n=1) and histidine (n=1). When related to the FLT3 length mutations
- 36 -
which were found in 27/60 (45%) of patients, both genetic alterations occurred
independently and none of the patients carried both mutations.
6.2 A new and recurrent mutation of FLT3: FLT3-840GS
6.2.1 Detection of the FLT3-LM in exon 20
We used a PCR based method to screen for mutations of codons D835/836 in
patients with AML. After gel electrophoresis of the amplification products from
exon 20 we observed an additional PCR fragment which was slightly larger than
the PCR product of the wildtype allele in two patients (Figure 3).
Figure 3: Detection of the FLT3-LM in exon 20 in AML after agarose gel electrophoresis. Detection of the length mutation in exon 20 after conventional agarose gel electrophoresis of PCR-products from cDNA and genomic DNA. M: Molecular weight standard, H20: water control, C: Patient without exon 20 mutation, P1/2: patient 1/2 with the length mutation in exon 20.
RT-PCR was repeated more than ten times with different aliquots of RNA as well
as cDNA and always gave the same results. Sequencing revealed an insertion of
6 nucleotides between the codons S840 and N841 just five amino acids (AA)
downstream of D835. These nucleotides generated a BamHI restriction site which
was confirmed by BamHI digestion and gel electrophorese. For this purpose, the
TKD2 of FLT3 was amplified by PCR, than PCR product was digested with BamHI
and detected on the gel (Figure 4). The DNA from FLT3-WT was used as a
control.
- 37 -
Figure 4: Confirmation of the mutation by BamHI digestion after polyacrylamide gel electrophoresis. M: molecular weight standard, MT: patient with mutation, WT: two patients without mutation, H20: water control. The PCR products were digested with BamHI and detected after polyacrylamid gel electrophoresis.
The Figure 5 shows the schematic presentation of the FLT3-840GS mutation.
Figure 5: Schematic presentation of the FLT3-840GS mutation. WT- FLT3 wild type; MT- mutation; TM- transmembrane domain; JM-juxtamembrane domain; PTK1 and 2 – protein tyrosine kinase domain 1 and 2, respectively; KI- kinase insert.
For patient 1 a DNA sample was available and the mutation could be shown also
at the genomic level by PCR, confirming the presence of the mutation (Figure 4).
The respective mutation was not detected in further 357 unselected AML pts.
- 38 -
Additional screening for frequent genetic mutations in these two patients showed
no further activating mutations of FLT3, NRAS, KIT or MLL (Table 4).
Table 4: Clinical data and alterations of other molecular markers in the two patients carrying the FLT3-840GS mutation. FLT3-LM, length mutations in the JM-region of FLT3; FLT3D835, mutations in codons 835/836; KIT D816, point mutations in codon 816; NRAS, activating point mutations in codons 12, 13 and 61; MLL-PTD, MLL-partial tandem duplication.
6.2.2 Generation of FLT3-840GS Ba/F3 cell lines
To analyze the transforming potential of the FLT3-840GS mutant, Ba/F3 FLT3-
840GS, FLT3-WT, FLT3-ITD and mock-expressing cells were generated. Ba/F3
cells were transduced with the pMSCV-IRES-EYFP retroviral expression vector
that contains an IRES site and allows translation of EYFP and the FLT3 construct
from the same transcript. Transduced EYFP-positive cells were sorted by FACS
on the basis of EYFP fluorescence and were expanded in the presence of IL-3. As
a positive control we generated Ba/F3 cells expressing the FLT3-ITD (W51)
mutant. (Spiekermann, Dirschinger et al. 2003)
As shown in Figure 6, the FLT3-840GS expressing Ba/F3 cells grew factor-
independently, whereas mock-expressing cells were unable to proliferate in the
absence of IL-3.
- 39 -
Figure 6: Transforming potential of the FLT3-840GS mutant. Ba/F3 cells transduced with either pMSCV-EYFP-IRES-FLT3-840GS or empty vector (pMSCV-EYFP-IRES, mock) were grown in the absence or presence of IL-3 as indicated. Results represent means ± SD of three independent experiments.
6.2.3 FLT3 expression and hyperphosphorylation in FLT3-840GS transformed
Ba/F3 cells.
To confirm the identical expression level of all FLT3 constructs flow cytometry
analysis was performed. For this purpose, FLT3WT, FLT3ITD, FLT3-840GS or empty
vector expressing Ba/F3 cells were incubated with a mouse isotype-matched control
antibody (open histograms) or CD135-phycoerythrin (filled histograms) antibody. Viable
cells were analyzed using a FacsCalibur flow cytometer (Figure 7).
Figure 7: FLT3 expression in Ba/F3 MIY (vector), FLT3-WT, FLT3-ITD or FLT3-840GS expressing cells.
- 40 -
Given the profound biological differences between the FLT3-WT and FLT3-MT (TKD or
LM) constructs, we next analyzed the FLT3 receptor activation. Lysates from Ba/F3 cells
were immunoprecipitated with αFLT3-antibody and analyzed by Western blot using an
antiphosphotyrosine (αPY) antibody, stripped and reblotted with αFLT3 antibody. Our
data show, that the FLT3-840GS and the FLT3-ITD receptors were hyperphosphorylated
in contrast to the FLT3-WT receptor (Figure 8).
Figure 8: The FLT3-840GS mutant receptor is hyperphosphorylated in Ba/F3 cells.
6.2.4 Transforming potential of the FLT3-840GS mutant The detailed analysis of the growth characteristics of FLT3-840GS Ba/F3 cells showed a
significantly slower growth rate compared to the FLT3ITD expressing cells. To analyze,
whether Ba/F3 cells expressing FLT3-840GS can achieve a maximum proliferation rate,
the cells were grown for 72h in the absence or presence of different concentrations of IL-3
as indicated. Ba/F3 cells expressing FLT3-WT or FLT3-ITD were used as a control. The
growth of FLT3ITD expressing Ba/F3 cells at 72h was defined as 100%. Our data clearly
show that for maximal proliferation, the FLT3-840GS expressing cells required additional
IL-3 at concentrations as low as 0.1 ng/ml (Figure 9).
- 41 -
Figure 9: For maximal proliferation the FLT3-840GS expressing cells require additional IL-3.
Results represent means ± SEM of three independent experiments.
6.2.5 FLT3-840GS activates the MAPK-STAT signalling pathway of FLT3
To identify common signalling pathways that specifically transduce the
transforming signals of the FLT3-840GS- mutated receptor, we analyzed the
activity of this mutant to activate STAT5 and MAPK. Using phosphospecific
antibodies, we found that FLT3-840GS induced a strong activation of MAPK
(Figure 10A), but only a weak activation of STAT5 (Figure 10B).
6.2.6 Sensitivity of the new mutation FLT3-840GS to the selective PTK
inhibitor SU5614 We next asked whether the FLT3-840GS mutant is sensitive to the growth
inhibitory activity of SU5614, a FLT3 PTK inhibitor. (Spiekermann, Dirschinger et
al. 2003) Our results clearly show that SU5614 induced a growth inhibition of the
FLT3-840GS as well as of the FLT3ITD transformed Ba/F3 lines in the absence,
but not in the presence of IL-3 (Figure 11).
- 42 -
Figure 10: Activation of FLT3 downstream targets (STAT5 and MAPK) in Ba/F3 cells transformed with FLT3-ITD and FLT3-840GS mutants. A. Lysates were prepared from Ba/F3 cells expressing mock, FLT3-WT, FLT3-ITD, FLT3-840GS constructs after IL-3 stimulation (for 5 min, RT) and without stimulation (+ and -). Immunoblotting of lysates was performed with anti-pMAPK and anti-MAPK antibodies. B. Lysates were prepared from Ba/F3 cells expressing mock, FLT3-WT, FLT3-ITD, FLT3-840GS constructs. Immunoblotting of lysates was performed with anti-pSTAT5 and anti-STAT5 antibodies.
Figure 11: Sensitivity of the FLT3-840GS mutant to the growth inhibitory activity of the FLT3 PTK inhibitor SU5614 Dose-responses curves of the inhibitory activity of SU5614 in Ba/F3 FLT3-ITD and FLT3-840GS cells after 72h of incubation. Ba/F3 cells expressing different FLT3-constructs were seeded at a density of 4x104 cells/ml in the absence or presence of different concentrations of SU5614. Viable cells were counted after 72 h by trypan blue exclusion.
- 43 -
6.3 A new point mutation in the juxtamembrane region of the FLT3: FLT3-V592A 6.3.1 MM1 and MM6 cell lines
MonoMac1 and MonoMac6 cell lines were established from the peripheral blood of
a 64-year-old man with leukemia (AML FAB M5) (DSMZ). Previous studies have
shown that these cell lines express an autophosphorylated FLT3 receptor
sensitive to SU5614. (Spiekermann, Dirschinger et al. 2003)
To further clarify the mechanisms of constitutive FLT3 activation in MM6 and MM1
cells, the complete FLT3 cDNA was sequenced. Both MM1 and MM6 cells carry a
point mutation in the JM region, which result in the substitution of valine by alanine
at position 592 (V592A) (Figure 12).
Figure 12: Schematic presentation of the FLT3-V592A mutation.
6.3.2 Generation of FLT3-V592A expressing Ba/F3 cells
To analyze the transforming potential of the FLT3-V592A mutation, Ba/F3 FLT3-
V592A cells were generated. Overexpression of this mutant in IL-3-dependent
Ba/F3 cell lines resulted in factor-independent growth, thereby showing the
transforming activity of this mutant (Figure 13). Compared with FLT3-ITD-
expressing cells, FLT3-V592A-Ba/F3 cells showed a slightly slower growth rate.
- 44 -
Figure 13: Ba/F3 cells transduced with either the pMSCV-EYFP-IRES-FLT3-WT/ITD or V592A construct were grown in the absence or presence of IL-3 as indicated. Results represent means ± SD of three independent experiments.
6.3.3 Sensitivity of the new mutation FLT3-V592A to the selective PTK
inhibitor SU5614
The selective PTK inhibitor SU5614 has been reported to inhibit the growth of
FLT3-ITD and FLT3-D835Y carrying Ba/F3 cells. (Yee, O'Farrell et al. 2002;
Spiekermann, Dirschinger et al. 2003)To analyze the sensitivity of the FLT3-
V592A mutant to SU5614, FLT3-V592A expressing Ba/F3 cells were cultured in
the presence of different concentrations of SU5614 for 72 hours. FLT3-V592A
and FLT3-ITD showed a similar and high sensitivity to SU5614 (IC50 =0.1) (data
not shown).
6.3.4 Importance of the Y589/591 tyrosine residues in FLT3-V592A signalling
FLT3 is constitutively activated by the substitution of Ala for Val at codon 592 in
the juxtamembrane domain, and this mutation confers a factor independent growth
on IL-3-dependent cell line. To examine how FLT3-V592A yields oncogenic signal
- 45 -
transduction, we constructed the expression vector pMSCV-YEFP carrying FLT3
mutants that encode the Y→F substituted FLT3-V592A or FLT3-D835Y. To
examine further the effect of Y→F substitution at codons 589 and 591 on the
factor-independent growth by FLT3-V592A and FLT3-D835Y, these constructs
were transfected into IL-3 dependent Ba/F3 cell lines. As controls, Ba/F3 cells
were also transfected with the expression vector carrying FLT3-V592A and FLT3-
D835Y.
As shown in Figure 14A, Ba/F3 FLT3-D835Y-Y589/591F cells showed factor-
independent growth at an almost similar level to Ba/F3 FLT3-D835Y cells.
Ba/F3 FLT3-V592A Y589/591F cells also proliferated in a factor-independent
manner, but their magnitude of proliferation was lower than that of Ba/F3 FLT3-
V592A cells (Figure 14B).
- 46 -
Figure 14: Ba/F3 cells transduced with either pMSCV-EYFP-IRES-FLT3-D835Y, D835Y-Y589/591F (A) or V592A, V592A-Y589/591F (B) were grown in the absence or presence of IL-3 as indicated. Results represent means ± SD of three independent experiments.
These findings indicate that tyrosines at codons 589 and 591 are important for full
transformation activity of the FLT3-V592A, but not of the FLT3-D835Y mutant
receptor.
6.4 Sensitivity of the different FLT3-TKD point mutations to the PTK inhibitor SU5614 6.4.1 The FLT3-TKD mutants D835Y/V/H/Q induce IL-3 independent growth in
Ba/F3 cells
In order to analyze the transforming potential of FLT3-TKD mutants in IL-3
dependent Ba/F3 cells, we generated FLT3-D835 Y/V/H/Q expressing cell lines.
Ba/F3 cells were transduced with the pMSCV-IRES-EYFP retroviral expression
vector that contains an IRES site and allows translation of EYFP and the FLT3
construct from the same transcript. Transduced EYFP-positive cells were sorted
by FACS on the basis of EYFP fluorescence and were expanded in the presence
of IL-3. Identical expression level of all FLT3 constructs was confirmed by flow
cytometry and by Western blot after immunoprecipitation (IP) with a FLT3 specific
antibody (data not shown). As a positive control we generated Ba/F3 cells
expressing the FLT3-ITD (W51) mutant. (Spiekermann, Dirschinger et al. 2003)
As described previously, the FLT3-ITD, but not the FLT3-WT construct conferred
IL-3 independent growth. All FLT3-D835 mutant-transduced Ba/F3 cell lines were
able to grow in the absence of IL-3 at a growth rate comparable to that of the
FLT3-ITD expressing cells (Figure 15).
- 47 -
Figure 15: Ba/F3 cells transduced with either pMSCV-EYFP-IRES-FLT3-WT/ITD or D835H/Q/V/Y were grown in the absence or presence of IL-3 as indicated. Results represent means ± SD of three independent experiments.
6.4.2 FLT3-TKD mutants activate the STAT5 and MAPK signaling pathways We and others have previously shown that FLT3-ITD and TKD mutants induce a
constitutive STAT5 and MAPK activation in Ba/F3 or 32D cells. (Hayakawa,
Towatari et al. 2000; Mizuki, Fenski et al. 2000; Tse, Mukherjee et al. 2000; Tse,
Novelli et al. 2001; Spiekermann, Dirschinger et al. 2003)To characterize the
signaling properties of FLT3-TKD mutants, we analyzed lysates from Ba/F3 cells
expressing the FLT3-WT, -ITD or -D835Y/V/H/Q constructs. Using an anti-
phosphotyrosine antibody, we could clearly show that the FLT3-D835Y/V/H/Q-
transduced Ba/F3 cells express a hyperphosphorylated FLT3 receptor (data not
- 48 -
shown). Furthermore, we analyzed the ability of FLT3-TKD mutants to activate
STAT5 and MAPK, two known downstream targets of FLT3-ITD mutants. For this
purpose, crude lysates from Ba/F3 cells expressing the FLT3-D835Y/V/H/Q
mutants were analyzed by phosphospecific antibodies against STAT5 and MAPK.
As shown in Figure 16 all FLT3-D835 mutants induced a strong constitutive
activation of STAT5 and MAPK at levels comparable to the FLT3-ITD construct.
Figure 16: Activation of FLT3 downstream targets (STAT5 and MAPK) in Ba/F3 cells transformed with FLT3-ITD and TKD mutants Lysates were prepared from Ba/F3 cells expressing the FLT3-WT, FLT3-ITD, FLT3-D835H/Q/V/Y constructs. Immunoblotting of lysates was performed with anti-pSTAT5 and anti-STAT5 (A), anti-pMAPK and anti-MAPK antibodies (B). These data clearly demonstrate that all FLT3-TKD mutants analyzed in our study
have transforming potential and induce a hyperphosphorylation of FLT3. STAT5
and MAPK represent common downstream targets of the constitutively active
FLT3-ITD and FLT3-TKD-mutant receptors.
- 49 -
6.4.3 FLT3-TKD mutants differ significantly in their sensitivity to the growth inhibitory activity of the FLT3 PTK inhibitor SU5614 We and others have previously reported that the PTK inhibitor SU5614 inhibits the
growth of FLT3-ITD and FLT3-D835Y carrying Ba/F3 cells. (Yee, O'Farrell et al.
2002; Spiekermann, Dirschinger et al. 2003)To analyze the sensitivity of different
FLT3-D835 mutants to SU5614 we cultured FLT3- D835Y/V/H/Q-expressing
Ba/F3 cells in the presence of different concentrations of SU5614 for 72 hours.
Ba/F3 cells expressing different FLT3-constructs were seeded at a density of
4x104 cells/ml in the absence or presence of different concentrations of SU5614.
Viable cells were counted after 72 h by trypan blue exclusion. As shown in Figure
17A, FLT3-TKD mutants differ significantly in their sensitivity to the inhibitor. FLT3-
D835Y and FLT3-ITD cells show a similar and high sensitivity to SU5614
(IC50=0.2µM and 0.1µM respectively).
Figure 17: FLT3-TKD mutants differ significantly in their sensitivity to the growth inhibitory activity of the FLT3 PTK inhibitor SU5614 A. Dose-responses curves of the inhibitory activity of SU5614 in Ba/F3 FLT3-ITD and FLT3-TKD cells after 72h of incubation. The growth of cells that were incubated without inhibitor was defined as 100%. Values represent means and standard errors from three independent experiments. B. Sensitivity of FLT3-ITD and TKD mutants to SU5614. Values represent means and standard errors from three independent experiments.
- 50 -
In contrast, the FLT3-D835Q and D835V mutants were significantly less sensitive
to the inhibitor (IC50=0.4 and 1µM respectively), and the FLT3-D835H was the
most resistant mutant (IC50>10µM). At concentrations of 1µM of inhibitor FLT3-ITD
cells showed a viability of 3% compared to 10% (FLT3-D835Y), 38% (FLT3-
D835Q), 52% (FLT3-D835V) and 78% (FLT3-D835H) after 72 hours of incubation
(Figure 17B).
These data demonstrate that FLT3-TKD mutants differ significantly in their
sensitivity to the FLT3 PTK inhibitor SU5614 in the following order: D835Y >
D835Q > D835V > D835H.
6.4.4 SU5614 induces apoptosis in FLT3-ITD and FLT3-D835Y, but not in FLT3-D835H transformed cells
To further characterize the mechanisms of primary resistance of the FLT3-D835H
mutant to SU5614, we analyzed induction of apoptosis after inhibitor treatment of
FLT3-ITD, D835Y and D835H transformed Ba/F3 cells with SU5614.
Figure 18A (upper panel) shows that FLT3-ITD transformed cells underwent rapid
apoptotic cell death after exposure to increasing concentration of SU5614 after 24
hours as measured by the expression of annexin V/7-AAD. FLT3-D835Y cells
show the same level of sensitivity to SU5614 (IC50 = 0.1µM) (Figure 18A, middle
panel), but not the FLT3-D835H cells (Figure 18A, bottom panel). In the presence
of 5µM of SU5614 FLT3-D835H cells show only 18% apoptotic cells in comparison
to FLT3-ITD (63%) and FLT3-D835Y cells (60%). A representative experiment is
shown in Figure 18B and clearly demonstrates that SU5614 at a concentration of
5µM induces apoptosis in 11.6% of D835H (lowest panel) cells compared to
60.2% and 78.3% in FLT3-D835Y (middle panel) and FLT3-ITD (upper panel)
expressing Ba/F3 cells, respectively.
- 51 -
Figure 18: SU5614 induces apoptosis in FLT3-ITD and FLT3-D835Q/V/Y, but not in FLT3-D835H cells. A. and B. Ba/F3 cells transduced with the FLT3-ITD, FLT3-D835Y or FLT3-D835H constructs were incubated with different concentrations of SU5614 for 24 hours and were analyzed by flow cytometry after staining with annexin V-PE and 7-AAD. Representative dot plots from one out of three independently performed experiments are shown.
6.4.5 The FLT3 PTK inhibitor SU5614 downregulates autophosphorylation of the FLT3-ITD, but not the FLT3-D835H receptor mutant It was recently shown that SU5614 inhibits phosphorylation of the FLT3-ITD
receptor and downregulates STAT5 and MAPK phosphorylation. (Yee, O'Farrell et
al. 2002; Spiekermann, Dirschinger et al. 2003)To confirm the resistance of FLT3-
D835H cells to SU5614 on the level of individual signaling pathways we analyzed
the activation of FLT3, MAPK and STAT5 after incubation with SU5614.
For this purpose, 293T cells were transiently transfected with either the FLT3-ITD
or the D835H construct. Lysates were prepared after incubation of the cells with
SU5614 (0, 1, and 10µM) for 4 hours and immunoprecipitated using a polyclonal
anti-FLT3 antibody. The phosphorylation of FLT3 was analyzed by Western blot
analysis using a monoclonal anti-phosphotyrosine (α-PY) antibody. As shown in
- 52 -
Figure 19A, SU5614 induced an efficient dephosphorylation of the FLT3-ITD
receptor at a concentration of 1µM (38.5% of control, defined as the FLT3
phosphorylation in the absence of SU5614 compared to basal levels of FLT3
measured by densitometry). In contrast, the D835H mutant was still strongly
hyperphosphorylated (92.7%) in the presence of 1µM SU5614. At concentrations
of 10µM SU5614 the FLT3-ITD construct was almost completely
dephosphorylated (7.4%), whereas a significant autophosphorylation was still
detectable in FLT3-D835H transfected cells (46.4%).
We further analyzed the effects of SU5614 on the activation of two important
downstream signalling pathways activated by FLT3-LM/TKD mutants. In the
presence of 1µM of SU5614 STAT5 phosphorylation in Ba/F3 FLT3-ITD cells was
almost completely inhibited (Figure 19B). In contrast, in Ba/F3 FLT3-D835H
expressing cells STAT5 was only slightly dephosphorylated in the presence of this
concentration of inhibitor. The incubation of the cells with 10µM of SU5614
produced an almost complete inhibition of STAT5 phosphorylation in both Ba/F3
FLT3-ITD and D835H transduced cell lines.
Figure 19: The FLT3 PTK inhibitor SU5614 downregulates autophosphorylation of the FLT3-ITD, but not FLT3-D835H receptor mutants
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A. Tyrosine phosphorylation of FLT3 was determined by Western blot analysis using a monoclonal anti-PY antibody and identical loading was confirmed by reblotting with a polyclonal anti-FLT3 antibody. B. The expression and phosphorylation of STAT5 in Ba/F3 MIY, FLT3-ITD and FLT3-D835H expressing cells treated with 0.1, 1 and 10µM of SU5614 or untreated cells was determined by Western blot analysis.
These results confirm at a molecular level the different sensitivity of FLT3-TKD mutants to FLT3 PTK inhibitors.
6.5 Mechanisms of drug resistance to SU5614
6.5.1 Generation of SU5614-resistant Ba/F3 FLT3-ITD cells FLT3 PTK inhibitors are now being evaluated in clinical phase I/II studies in
patients with AML (see above), but it is unknown to what extent clinical resistance
will develop and influence the clinical efficiency of these inhibitors.
Figure 20: Generation of SU5614-resistent cell lines. A. The polyclonal Ba/F3 FLT3-ITD cell line was cultivated in RPMI-1640 medium containing 10% FBS in the presence of increasing concentrations of SU5614 B. The Ba/F3 FLT3-ITD-R4 cells were seeded at a density of 4x104 cells/ml in the absence and presence of different concentrations of SU5614 and viable cells were counted after 72 hours by trypan blue exclusion.
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To address this important issue, FLT3-ITD-W51 expressing polyclonal Ba/F3 cells
were cultivated either in the presence of increasing concentrations of SU5614
(0.2µM to 0.8µM) or in the absence of inhibitor (control cells) for a time period of 3
months (Figure 20A). After a cultivation period of 2 weeks at a concentration of
0.2µM SU5614 two independent cell lines were obtained that were each split in
two sublines (ITD-R1+R2 and ITD-R3+R4, respectively). In contrast to the control
cell line all four cell lines (ITD-R1-R4) growing in the presence of inhibitor
developed a partial resistance to SU5614 and showed an IC50, which was at least
7-fold higher than the IC50 of control Ba/F3 FLT3-ITD cells (Table 5).
A representative example is shown in Figure 20B: Ba/F3 FLT3-ITD-R4 cells were
cultured for 72 hours in the presence of increasing concentration of SU5614. The
IC50 for SU5614 was 0.2µM in the control ITD cell line and 1.3 µM in ITD-R4 cells.
At high concentrations of 10µM of inhibitor the ITD-R4 line was still sensitive to the
growth inhibitory activity of SU5614 showing that the proliferation of these cells
was still dependent on the presence of the activating FLT3 mutant.
Table 5: Functional and molecular characterization of Ba/F3 FLT3-ITD-R1-4 cells.
The IC50 of SU5614 and Ara-C in ITD-R1-4 cells was determined as described in Figure
20. FLT3 expression was measured by FACS and the mean channel fluorescence (MCF)
was calculated. The expression of FLT3 (WB) was determined by Western blot analysis
using an anti-FLT3 antibody. The blot was stripped and reprobed with an anti-β-actin
antibody. The results were quantified by densitometry according to the FLT3-ITD/β-actin
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ratio of parental cells that was set to 100%. FLT3-TKD mutation indicates the presence
(Y842H and D835N) (or absence (-)) of point mutations in tyrosine kinase domain (TKD).
6.5.2 Activation of STAT5 and MAPK in FLT3-ITD-R1-4 cell lines in response
to SU5614 To confirm the resistance to SU5614 at a molecular level, we analyzed two
important downstream targets of FLT3: STAT5 and MAPK. For this purpose FLT3-
ITD-R2 and R4 cell lines were incubated with different concentrations of SU5614
(0, 1 and 10µM) for 4 hours. As shown in Figure 21, SU5614 efficiently induces
dephosphorylation of STAT5 and MAPK at a concentration of 1µM in Ba/F3 FLT3-
ITD cells. In contrast to the parental cell line, STAT5 and MAPK were still
phosphorylated in the FLT3-ITD-R2 and R4 cells even in presence of 10µM
SU5614. These results are in good agreement with the IC50 of these cell lines in
cell proliferation assays showing a 4 fold higher IC50 of ITD-R2 compared to ITD-
R4 cells (see Table 5).
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Figure 21: Activation of STAT5 and MAPK in FLT3-ITD-R1-4 cell lines in response to SU5614. The phosphorylation status of STAT5 and MAPK in extracts of Ba/F3 FLT3-ITD and FLT3-ITD-R4/2 cells treated with 1 and 10 µM of SU5614 was determined by Western-Blot analysis using the polyclonal anti-pSTAT5 (A.) and anti-pMAPK antibodies (B.). Expression of STAT5 and MAPK in the same lysates was analyzed by immunoblotting with polyclonal anti-STAT5 and anti-MAPK antibodies.
6.5.3 Ba/F3 FLT3-ITD-R1-4 cells are resistant to the FLT3 PTK inhibitor AG1295, but not to Genistein and Ara-C To further characterize the SU5614-resistant cell lines and analyze whether these
cells developed a specific resistance to FLT3 PTK inhibitors, we cultivated FLT3-
ITD-R1-4 cells in the presence of two different PTK inhibitors: either AG1295, that
has inhibitory activity to class III PTK (Levis, Tse et al. 2001) or the broad
spectrum PTK inhibitor Genistein. (Akiyama, Ishida et al. 1987) We could
demonstrate that all SU5614-resistant cell lines developed also resistance to the
FLT3 selective PTK inhibitor AG1295 (Figure 22A,B), but not to Genistein (Figure
22C).
Further we investigated whether the ITD-R1-4 cells developed an unspecific
resistance to apoptotic cell death induced by cytotoxic drugs. For this purpose we
exposed these cells to different concentrations of Ara-C (cytosine arabinoside, 0-5
µg/ml), a deoxycytidine analogue that is the most effective cytotoxic agent in the
treatment of AML. We found that FLT3-ITD-R1-4 mutants are sensitive to Ara-C at
a level comparable to that seen in the parental FLT3-ITD line (Figure 22B,
summarized in Table 5).
These data clearly show that the ITD-R1-4 cell lines are resistant to the growth
inhibitor activity of the FLT3 PTK inhibitor AG1295, but not to unselective PTK
inhibitors or the cytotoxic agent Ara-C.
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Figure 22: Ba/F3 ITD-R1-4 cells are resistant to AG1295, but not to Genistein and Ara-C. A. Ba/F3 FLT3-ITD-R1-4 cells were seeded in the absence or presence of 5µM AG1295 and counted after 48 hours by trypan blue exclusion. B. Parental Ba/F3 FLT3-ITD and FLT3-ITD-R4 cells were cultured for 48 hours in the absence of IL-3 with different concentrations of Ara-C. C. Parental Ba/F3 FLT3-ITD and FLT3-ITD-R4 cells were cultured for 72 hours in the absence of IL-3 with different concentrations of Genistein (as indicated).
6.5.4 Expression of FLT3 protein in resistant cell lines
Overexpression of the target kinase is a well known mechanism of PTK-inhibitor-
resistance. (le Coutre, Tassi et al. 2000; Mahon, Deininger et al. 2000; Weisberg
and Griffin 2000)To further evaluate the mechanisms of SU5614 resistance in the
ITD-R1-4 cell lines we analyzed FLT3 surface expression by flow cytometry using
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a PE-labelled monoclonal anti-FLT3 antibody. All resistant cell lines expressed
significantly higher levels of FLT3 in comparison to the parental FLT3-ITD cell line
(Figure 23, Table 5).
These data were confirmed by Western blot analyses using a polyclonal antibody
directed against the interkinase domain of FLT3. Identical protein loading in all
lanes was confirmed by immunoblotting using an anti-ß-actin antibody. FLT3
expression was significantly higher in ITD-R4- (1285%), ITD-R3- (1016%), ITD-
R2- (918%) and ITD-R1-cells (725%) compared to FLT3 levels in the parental
FLT3-ITD cell line (100%).
Figure 23: Expression of FLT3 in SU5614-resistent cell lines in comparison to parental FLT3-ITD cells. A. The expression of FLT3 in Ba/F3 FLT3-ITD-R1-4 was analyzed by FACS analysis. Open histograms represent isotype control (PE-labelled control antibody); filled histograms show fluorescence intensity of CD135. B. FLT3 expression was determined by immunoblotting using a polyclonal anti-FLT3 antibody.
6.5.5 The SU5614 resistant ITD-R1-4 cell lines acquired distinct FLT3-TKD mutations
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Another mechanism of clinical resistance to PTK inhibitors represents the
acquisition of distinct mutations in the ATP-binding site and the kinase activation
loop. This phenomenon was first described in patients with CML who developed
clinical resistance to imatinib and were found to carry imatinib-resistant point
mutants of BCR-ABL in their leukemic blasts. (Gorre, Mohammed et al. 2001;
Branford, Rudzki et al. 2002; Hofmann, Jones et al. 2002; Roumiantsev, Shah et
al. 2002; Shah, Nicoll et al. 2002; von Bubnoff, Schneller et al. 2002)We
hypothesized that similar mechanisms might also be responsible for resistance to
FLT3 PTK-inhibitors in SU5614-resistant FLT3-ITD cell lines. For this purpose we
analyzed the known hot spot regions for activating mutations in the FLT3 gene
found in patients with AML: the juxtamembrane region (JM) and the activation loop
of the kinase domain. The JM region was screened for mutations using the
previously published primers 11F and 12R and subsequent visualization of the
PCR-products in agarose gels. (Kiyoi, Naoe et al. 1997; Schnittger, Schoch et al.
2001)The ITD-R1-4 lines expressed the parental ITD (W51) mutation, as shown by
an identical size of the PCR product compared with the control cell line (Figure
24A).
To screen for mutations in the TKD domain we used melting curve analyses after
amplification of a 244 base pair fragment by real-time PCR as described
previously. (Schnittger, Schoch et al. 2001; Schnittger, Boell et al. 2002)The
design of the hybridization probe allows detection of the point mutations and
insertions/deletions ranging from amino acid 825 to 839 in the TKD domain of
FLT3. The real-time PCR analysis demonstrated abnormal melting curves for the
ITD-R3 and R4 mutants using either cDNA or genomic DNA as a template.
Nucleotide sequencing of genomic DNA from these cells confirmed a single and
identical G→A nucleotide (nt) exchange in both the ITD-R3 and ITD-R4 cell lines
resulting in a D835N mutation. Sequential analysis of the FLT3-ITD-R4 cell line at
12 weeks and 18 weeks during selection with SU5614 indicated that the ratio of
D835N/D835 DNA continuously increased over time (Figure 24B). The melting
curve analyses shown in Figure 9B clearly demonstrates that the D835N mutant
allele accounted for approximately 50% of DNA after 12 weeks of selection and
increased to 100% of total DNA after continuous exposure to SU5614 for another
6 weeks. These results were confirmed by direct nucleotide sequencing of the
DNA templates used for melting curve analysis (data not shown).
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Figure 24: Molecular characterization of SU5614-resistant cells. A. The juxtamembrane region of FLT3 was amplified by PCR using the published primer pair (11F and 12R) (Kiyoi, Naoe et al. 1997; Schnittger, Schoch et al. 2001)and amplification products were separated by agarose gel electrophoresis and viewed under UV illumination after ethidium bromide staining. M: DNA molecular weight marker, H2O: water control, Ba/F3: native Ba/F3 cells, WT: Ba/F3 FLT3-WT, ITD: Ba/F3 FLT3-ITD, ITD-R1-4: Ba/F3 FLT3-ITD-R1-4, PP: positive control (AML patient carrying a FLT3-LM). B. Detection of FLT3-TKD mutations was performed by melting curve analysis after amplification of a 244 bp fragment by real time PCR as described previously. (Schnittger,
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Schoch et al. 2001; Schnittger, Boell et al. 2002)In the presence of the FLT3-TKD wild type sequence or the FLT3 TKD mutant DNA the fluorescence peak is observed at 63°C and 55°C, respectively. C. The structural domains of the FLT3 protein with the position of the TKD mutations found in Ba/F3 FLT3-ITD-R1-4 cells (D835N and Y842H) are shown.
After the identification of a D835N mutant in ITD-R3 and R4 cells, we sequenced
the entire TKD domain of the ITD-R1 and R2 cell lines to screen for further
mutations outside the region that was covered by the hybridization primers used in
the light cycler analysis. Direct nucleotide sequencing clearly demonstrated that
both the ITD-R1 and R2 cells carried a T→C nucleotide substitution in codon 842
of FLT3 resulting in an Y842H mutation (Figure 24C).
Taken together, these data clearly indicate that distinct mutations in the PTK
domain of FLT3 are acquired in Ba/F3 FLT3-ITD cells during selection with the
FLT3 PTK inhibitor SU5614.
6.6 “Dual” mutants FLT3 ITD-TKD are resistant to SU5614
6.6.1 The FLT3-ITD/TKD “dual” mutation is responsible for the SU5614 resistant phenotype in FLT3-ITD-R1-4 cells
To confirm that the D835N and Y842H TKD mutations are responsible for the
resistance to SU5614 in ITD-R1/2 and ITD-R3/4 cells, we introduced either the
D835N or the Y842H mutation in the FLT3-WT and the FLT3-ITD constructs.
Then Ba/F3 cell lines expressing the FLT3-D835N, Y842H or the FLT3-ITD mutant
alone were generated. In addition, the FLT3 “dual” mutant FLT3-ITD/D835N and
FLT3-ITD/Y842H Ba/F3 cell lines were established. All FLT3 mutants conferred IL-
3 independent growth to Ba/F3 cell lines (data not shown, summarized in Table 6).
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Table 6: Characterization of Ba/F3 cells expressing FLT3-ITD/TKD dual mutants. The IC50 of SU5614 in Ba/F3 cell lines expressing either the D835N or Y842H or the FLT3-ITD mutant alone and the FLT3-ITD/D835N, FLT3-ITD/Y842H “dual” mutants was determined as described in Figure 10.
As shown in Figure 25A, the IC50 of FLT3-D835N cells to SU5614 did not
significantly differ from the IC50 of FLT3-ITD cells (IC50 =0.1µM and 0.08µM
respectively). In contrast, the FLT3-ITD/D835N dual mutant cells were partially
resistant to SU5614 at levels comparable to the original ITD-R4 cell line (IC50=1.0
and 1.3µM respectively). Similar results were obtained when FLT3-Y842H and
FLT3-ITD/Y842H cell lines were analyzed (Figure 25B, Table 5).
We further analyzed whether the “dual” FLT3 ITD/TKD mutant provides a
competitive growth advantage to Ba/F3 cells in the absence or presence of 0.2µM
of SU5614 compared to the parental ITD cell line. For this purpose, we mixed MIG
FLT3-ITD (GFP+) and MIY FLT3-ITD/D835N (YFP+) expressing Ba/F3 cells in a
ratio of 10:1 and measured the percentage of GFP+ and YFP+ cells every 3-4 days
for a time period of 2 weeks by FACS analysis (Figure 26A,B).
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Figure 25: The FLT3-ITD/D835N and the FLT3-ITD/Y842H dual mutants restore the SU5614-resistant phenotype in Ba/F3 cells. (A.) The Ba/F3 FLT3-ITD, FLT3-D835N and FLT3-ITD/D835N, (B.) FLT3-Y842H and FLT3-ITD/Y842H expressing cells were seeded at a density of 4x104 cells/ml in the absence and presence of different concentrations of SU5614 and viable cells were counted after 72 hours by trypan blue exclusion. We could show that over a time period of 14 days the FLT3-ITD/D835N “dual”
mutant cells had a substantial competitive growth advantage in the presence, but
not in the absence of SU5614. The percentage of FLT3-ITD-D835N-YFP positive
cells in the presence of SU5614 increased from 10% (day 0) to 88% (day 14),
whereas the percentage of “dual” mutant YFP positive cells cultured in the
absence of SU5614 increased to only 22%.
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Figure 26: The FLT3-ITD/D835N dual mutant provides a competitive growth advantage to Ba/F3 cells in the presence of SU5614. A. and B. MIG FLT3-ITD (GFP+) and MIY FLT3-ITD/D835N (YFP+) expressing Ba/F3 cells were mixed in a ratio of 10:1 and the percentage of GFP+ and YFP+ cells was measured every 3-4 days for a time period of 2 weeks by FACS analysis in the presence (A.) or absence (B.) of 0.2µM of SU5614.
These data clearly show that the FLT3-ITD/TKD dual mutant restores the ITD-R1-
4 phenotype. Therefore, the acquisition of specific TKD mutations in the FLT3-ITD
gene represents a new mechanism of resistance to FLT3 PTK inhibitors in vitro.
6.6.2 “Dual” mutants are resistant to SU5614 independent from kind of mutation in juxtamembrane region of FLT3
We hypothesized, that various length of mutations in JM region can lead to
differences in sensitivity to SU5614. For this purpose we generated Ba/F3 cells
transformed with FLT3-V592A/D835N and FLT3-NPOS/D835N mutants. The
FLT3-NPOS mutant contains a 28-amino acid duplicated sequence inserted
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between amino acids 610/611 in contrast to FLT3-W51 mutant, which contains
only 6-amino acid duplicated sequence inserted between amino acids 601/602
and FLT3-V592A contains only point mutation.
Our results clearly demonstrate, that all of these dual mutants show partially
resistance to SU5614 although at a different degree (IC50=0.3µM for V592A-
D835N- only weak resistance and 1µM for NPOS-D835N - more strong) (Figure
27).
Figure 27: The FLT3-ITD/D835N and the FLT3-ITD/Y842H dual mutants restore the SU5614-resistant phenotype in Ba/F3 cells. (A.) The Ba/F3 FLT3-ITD, FLT3-D835N and FLT3-ITD/D835N, (B.) FLT3-Y842H and FLT3-ITD/Y842H expressing cells were seeded at a density of 4x104 cells/ml in the absence and presence of different concentrations of SU5614 and viable cells were counted after 72 hours by trypan blue exclusion. 6.6.3 The presence of FLT3-ITD and FLT3-TKD mutations on the two ‘‘alleles’’ do not confer SU5614 resistance
Furthermore, we analyzed whether the ITD and TKD mutations, which occurred on
the different alleles, also might lead to resistance to PTK-inhibitors. For this
purpose, we generated Ba/F3 cells transformed with FLT3-ITD//FLT3-D835N and
FLT3-ITD//FLT3-WT. As a control we generated Ba/F3 FLT3-ITD cell line. Our
data clearly show that occurrence of ITD and TKD mutations on two alleles is not
sufficient to induce resistance to PTK-inhibitor (Figure 28).
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Figure 28: The FLT3-ITD//FLT3-D835N and the FLT3-ITD//FLT3-WT mutants are completely sensitive to SU5614. The Ba/F3 FLT3-ITD, FLT3-ITD//FLT3-D835N and FLT3-ITD//FLT3-WT expressing cells were seeded at a density of 4x104 cells/ml in the absence and presence of different concentrations of SU5614 and viable cells were counted after 72 hours by trypan blue exclusion.
These data clearly confirm that occurrence of LM and TKD mutations on the same
allele, but not on two alleles lead to resistance to the PTK-inhibitor SU 5614.
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7. DISCUSSION 7.1 Functional characterization of two “new’’ FLT3 mutations in
AML: V592A and 840GS The data presented here clearly indicate that activating mutations in the FLT3
gene do not only occur within the juxtamembrane domain and in codons D835/836
but also in other positions of the A-loop of the catalytic domain. The A-loop
represents a hot spot region for activating mutations in class III RTK which have
been described for KIT (D816) and FLT3 (D835/836) (Yamamoto, Kiyoi et al.
2001)These mutations induce a conformational change of the A-loop which results
in the opening of the catalytic pocket and a constitutively active kinase activity.
Although no structural data on the catalytic domain of FLT3 are available which
would allow the detailed structure-function analysis, the close proximity to the AA
D835/836 suggests a similar mechanism of kinase activation by the FLT3-840GS
mutant.
Activating mutations of the FLT3 gene provide an essential anti-apoptotic and pro-
proliferative signal in primary AML cells and cell lines (Hayakawa, Towatari et al.
2000; Mizuki, Fenski et al. 2000; Tse, Mukherjee et al. 2000) Our results clearly
indicate that the FLT3840-GS mutant is hyperphosphorylated on tyrosine residues
and induces IL-3 independent growth in Ba/F3 cells. These in vitro data underline
the pathophysiologic role of this mutant for the leukemic phenotype in patients with
AML.
In order to identify downstream targets of FLT3-840GS mutant receptor, we
performed Western blot analysis with phosphospecific antibodies against STAT5
and MAPK. Our results confirmed strong MAPK activation by the FLT3-840GS, but
only weak STAT5 activation. We hypothesize the weak STAT5 activation
translates into a weak transforming activity of this mutation and used IL-3
stimulation to induce maximal proliferation activity. Our results show that IL-3
stimulation leads to an increased growth rate of the Ba/F3 FLT3-840GS cells in
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dose-dependent manner. Therefore, we suggest, that the FLT3-840GS mutant
requires additional mitogenic signal for full transformation. Possible explanation of
these results might be found in recently published article from Y.Minami et al,
where the authors show differences between anti-apoptotic pathways of wild-type
and mutated FLT3 receptors. The authors showed that STAT5 is the most
important downstream target for FLT3-ITD mutants, but the MAPK pathway is
indispensable for the FLT3-WT receptor. Based on the data shown here we
hypothesize that the FLT3-840GS mutant uses similar pathway compared to the
FLT3-WT receptor.
Another mutation that was found and described in this work is the FLT3-V592A.
This mutation represents the first point mutation in JM domain of FLT3. The
mutation was found in two AML cell lines (MM6 and MM1), but the frequency in
patients with AML is unknown. Probably the V592A represents rare mutation, but it
represents a good model for future investigations of FLT3 signalling. Recently it
was shown by Di Maio et al. that amino acid substitution in JM region of the
PDGFR led to activation of the receptor. V592 of FLT3 is also a highly conserved
amino acid in class III RTK (Rosnet, Marchetto et al. 1991)and further analysis will
be needed to investigate the mechanism of transformation by this mutation.
The data demonstrated here show that FLT3-V592A is an activating mutation,
which leads to hyperphosphorylation of the FLT3 receptor and induces IL-3-
independent growth in Ba/F3 cells. Our data also demonstrate that the FLT3-V592
mutant receptor activates two known downstream targets of FLT3: STAT5 and
MAPK. Furthermore, the specific FLT3 PTK inhibitor SU5614 was able to inhibit
IL-3-independent growth of MM6, MM1 (Spiekermann, Dirschinger et al. 2003)and
Ba/F3 FLT3-V592A cells.
In order to identify differences in FLT3-TKD and FLT3-V592A signalling, we
generated 589/591 Y→F substitution mutants of either FLT3-D835Y or FLT3-
V592A. Our data show that the tyrosine residues 589/591 play a crucial role for
transformation of Ba/F3 cells by the FLT3-V592A, but not by the FLT3-D835Y
mutant. These findings are in good agreement with results from Kyioi et al., which
found that these tyrosine residues are important for FLT3-ITD constructs (Kiyoi,
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Towatari et al. 1998) Although further studies are required, we suggest, that the
FLT3-V592A mutant and FLT3-ITD mutant activate similar signalling pathways.
Although the FLT3-840GS and FLT3-V592A are probably rare mutations, they
clearly show that activating mutations other than the FLT3-LM in the JM domain
and FLT3D835/836 in the kinase domain exist in patients with AML.
These findings are of significant clinical importance since activating FLT3
mutations could represent selective and specific molecular target structures for
therapeutical strategies using PTK inhibitors in AML (Levis, Tse et al. 2001; Tse,
Novelli et al. 2001)
7.2 FLT3 as a molecular target for selective therapeutic approaches in AML Our group and others have previously shown that selective FLT3 PTK inhibitors,
like SU5614, AG1295, PKC412 or CEP701 can induce growth arrest and
apoptosis in FLT3 transformed cell lines and in primary AML blasts (Levis, Tse et
al. 2001; Kelly, Yu et al. 2002; Weisberg, Boulton et al. 2002; Yee, O'Farrell et al.
2002; Spiekermann, Dirschinger et al. 2003) These promising in vitro results have
stimulated phase I/II clinical studies evaluating the efficacy of FLT3 PTK inhibitors
in patients with AML (Foran, Paquette et al. 2002; Smith, Levis et al. 2002; Stone,
Klimek et al. 2002)
In contrast to the well studied mechanisms of drug resistance found in patients
with BCR-ABL-positive CML and ALL treated with the PTK inhibitor imatinib, little
is known about primary and secondary mechanisms of resistance to FLT3 PTK
inhibitors. In the present study we could show that certain activating point
mutations found in the PTK domain of FLT3 in patients with AML significantly alter
the sensitivity to the FLT3 PTK inhibitor SU5614. As a mechanism of secondary
resistance we identified that FLT3-ITD transformed cell lines acquire distinct
mutations in the TKD domain after prolonged cultivation in the presence of FLT3
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PTK inhibitors. Reconstruction of these FLT3 “dual” mutants confirmed that the
presence of a certain TKD-mutant on the genetic background of the ITD mutation
was responsible for the FLT3-PTK inhibitor resistant phenotype. These data
provide genetic evidence that pre-existing and acquired mutations in the TKD
domain of FLT3 are sufficient to induce drug resistance to FLT3 PTK inhibitors.
Genetically heterogeneous mutations in the TKD domain of FLT3 have been found
in 7-8% of patients with AML and include point mutations at codon D835 and also
insertions/deletions in codons 835/836 (Abu-Duhier, Goodeve et al. 2001; Thiede,
Steudel et al. 2001; Yamamoto, Kiyoi et al. 2001; Thiede, Steudel et al. 2002) To
analyze the sensitivity of these mutants to FLT3 PTK inhibitors, we generated
Ba/F3 cell lines expressing the most frequent FLT3-TKD mutants, namely
D835Y/V/H. All FLT3-TKD mutants confer IL-3 independent growth to Ba/F3 cells
and activate similar signal transduction pathways (STAT5 and MAPK) compared
to the FLT3-ITD construct. Importantly, these FLT3-TKD mutants differed
significantly in their sensitivity to the growth inhibitory and apoptosis inducing
activity of the FLT3 PTK inhibitor SU5614 in the following order: FLT3-ITD = FLT3-
D835 Y > V > H.
AML-specific mutations in the TKD-domain of PTKs occur in conserved amino
acids at codons 835/836 and 816 in FLT3 and KIT, respectively (Abu-Duhier,
Goodeve et al. 2001; Yamamoto, Kiyoi et al. 2001; Ma, Zeng et al. 2002) It has
been hypothesized that these mutations change the conformation of the activation
loop (A-loop) thereby allowing spontaneous opening of the catalytic pocket and
ligand-independent activation of the kinase (Blume-Jensen and Hunter 2001;
Wybenga-Groot, Baskin et al. 2001) The conformational change in the A-loop of
KIT by the D816V mutations is probably also responsible for the resistance of this
mutant to the PTK inhibitor imatinib (Ma, Zeng et al. 2002; Zermati, De Sepulveda
et al. 2003) Based on these findings, the observation of decreased SU5614-
sensitivity of certain FLT3-TKD mutants, e.g. D835H might also be attributable to
the decreased affinity of the inhibitor for the catalytic pocket of FLT3. Recently,
Grundler et al. have analyzed the sensitivity of mutants of FLT3 at codon 835/836
to the FLT3 PTK inhibitors SU5614, PKC412 and AG1295. These authors found in
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accordance with our results that distinct FLT3-TKD mutations lead to an altered
sensitivity to PTK inhibitors (Grundler, Thiede et al. 2003)
These findings have important clinical implications for studies evaluating the
efficacy of FLT3 PTK inhibitors in patients with FLT3-TKD mutation positive AML.
FLT3-TKD mutations can change the affinity of the kinase to inhibitors and might
therefore result in clinical resistance. Treatment of AML patients carrying TKD
mutations will therefore require a preclinical sensitivity analysis to select an
appropriate inhibitor.
7.3 ‘’Dual’’ mutations in the FLT3 gene as a cause of drug resistance to FLT3 PTK inhibitors We characterized 4 sublines of the parental FLT3-ITD transformed Ba/F3 cell line
after selection in the presence of 0.2-0.8µM SU5614. All cell lines acquired a
partial resistance to the FLT3 PTK inhibitor SU5614, but not to other apoptosis
inducing agents like Ara-C, and showed an IC50 that was 10-25 fold higher
compared to that of control cells. Importantly, the ITD-R1-4 lines were completely
sensitive to SU5614 at high concentrations of 10µM of inhibitor showing that the
proliferation of all cell lines was still dependent on the presence of the FLT3
mutant. Additional experiments using the FLT3 PTK-inhibitor AG1295 clearly
showed that the SU5614-resistant phenotype induced a complete cross-resistance
with AG1295.
The detailed molecular analysis showed that each of the ITD-R1-4 cell lines
acquired a specific TKD mutation resulting either in a D835N or an Y842H FLT3
mutant. In sequential analyses performed in the ITD-R4 cell line during selection
with SU5614 the ratio of mutant/wild type DNA increased over time showing a
substantial competitive growth advantage of the ITD-D835N dual mutant
expressing cell population.
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Clinical samples obtained from patients with AML usually carry either the ITD or
the TKD mutation (Kottaridis, Gale et al. 2001; Thiede, Steudel et al. 2001;
Schnittger, Schoch et al. 2002; Thiede, Steudel et al. 2002) However, in some
patients both mutations can be detected and preliminary data suggest that these
patients might have an even worse clinical prognosis compared to patients with a
single ITD or TKD mutation (Karali, Dimitriadou et al. 2002; Moreno, Martin et al.
2003) In a recent study of 979 patients, Thiede et al. found an additional TKD
mutation in 17/200 (8.5%) of AML patients carrying a FLT3-LM. Further analysis
revealed that 40% of the LM and TKD mutations occurred on the same allele
showing that FLT3-LM/TKD “dual” mutants spontaneously arise in vivo (Thiede,
Steudel et al. 2002)Although the “dual” FLT3 LM/TKD mutant is infrequently found
at diagnosis in patients with AML, our in vitro data suggest that these mutants
might develop during treatment with FLT3 PTK inhibitors.
7.4 The FLT3-ITD/TKD dual mutation is responsible for the PTK inhibitor resistance. To prove that the acquisition of the TKD mutation is responsible for the FLT3 PTK
inhibitor resistance in the ITD-R1-4 cells we generated Ba/F3 cells expressing
either the FLT3-D835N/Y842H mutant alone or on the background of the ITD
mutation. Although both TKD mutants alone induced IL-3 independent growth in
Ba/F3 cells, these mutants were equally sensitive to SU5614 compared to the
FLT3-ITD-construct. In contrast, when introduced on the ITD background, both the
D835N and the Y842H mutation restored the SU5614 resistant phenotype in
Ba/F3 cells.
Although the FLT3-D835N and the Y842H mutants have transforming activity,
these constructs alone did not mediate resistance to SU5614. The explanation for
this phenomenon is not obvious, but one has to assume that only the combination
of the ITD and the D835N/Y842H mutation changes the conformation of the
catalytic domain in a way, which decreases its affinity for the FLT3 inhibitor.
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Structural analyses of the Ephrin-receptors have shown that both the activation
loop and the JM region directly interact with the PTK domain by the formation of
autoinhibitory loops (Blume-Jensen and Hunter 2001; Wybenga-Groot, Baskin et
al. 2001) To what extent the exact amino acid sequence and the site of insertion of
the FLT3-LM will influence the resistance to a specific FLT3 PTK inhibitor is
unknown. The high variability of FLT3-LM found in patients with AML must be
taken into account when choosing an appropriate FLT3 PTK inhibitor, as it is likely
that resistance or sensitivity to these compounds might also depend on the three
dimensional structure of the JM-region.
Although the FLT3-ITD/TKD dual mutant restores the SU5614 resistant phenotype
when introduced in FLT3-ITD transformed Ba/F3 cells, we cannot completely rule
out that other mechanisms might contribute to the phenotype of ITDR1-4 cells.
Such mechanisms of resistance include altered metabolism of the drug, induction
of drug efflux, e.g. by ABC transporters or overexpression of the target kinase.
Very recently, Weisberg et al. have generated a PKC412 resistant Ba/F3 cell line,
that was selected with increasing inhibitor concentrations and showed
overexpression of FLT3 as a potential mechanism of drug resistance (Weisberg,
Boulton et al. 2002) The detailed analysis of the ITD-R1-4 cell lines described here
showed overexpression of FLT3 protein. Although, the FLT3-ITD/TKD dual mutant
cells generated by transduction of Ba/F3 cells with the FLT3 cDNA carrying both
mutations resembled the Ba/F3 FLT3-ITD-R1-4 phenotype the IC50 of SU5614 in
ITD-R1-4 cells was still 2-5 times higher (see Table 5 and 6). It is possible that
overexpression of the FLT3 found in the ITD-R1-4 cells might contribute to the
enhanced SU5614-resistance in these cells compared to the “dual” ITD/TKD
mutant Ba/F3 cells. As shown in patients with CML developing clinical resistance
during imatinib treatment several mechanisms of resistance can occur in parallel
and factors, other than mutations in the gene, e.g. overexpression of the target
kinase are likely to contribute to drug resistance in vivo (Gambacorti-Passerini,
Barni et al. 2000; le Coutre, Tassi et al. 2000; Mahon, Deininger et al. 2000;
Weisberg and Griffin 2000).
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7.5 Different kind of mutations in the JM region of FLT3 induce FLT3 PTK inhibitor resistance in dual mutant receptors. To analyze whether different mutations in JM region of FLT3 together with different
TKD mutations would also confer resistance to FLT3 PTK inhibitors, Ba/F3 cell
lines transformed with different dual FLT3 mutants were generated: Ba/F3 FLT3
W51-D835N, Ba/F3 FLT3 NPOS-D835N, Ba/F3 FLT3 V592A-D835N and Ba/F3
FLT3 W51-D835Y. We hypothesized that a different length of mutation in JM
domain could probably lead to different conformation changes of the receptor and
in this way to different sensitivity to PTK inhibitor. Our data demonstrated that this
hypothesis was only partially right. Though these mutants differ in their sensitivity
to SU5614, all of them show partially resistance to SU5614. That means that
different kind of mutations in the JM region are able to induce PTK inhibitor
resistance in the FLT3 TKD background.
To clarify whether the two mutations have to occur on the same or on different
alleles to induce inhibitor resistance, we generated Ba/F3 cell lines transformed
with FLT3-ITD and FLT3-WT constructs and FLT3-ITD and FLT3-D835N mutants.
In agreement with our theory that ITD-TKD mutation lead to conformation
changes, the presence of the ITD and TKD mutations in different constructs was
insufficient to confer resistance to SU5614.
Taken together, mutations in the TKD domain of FLT3 can induce primary and
secondary resistance to FLT3 PTK inhibitors. These findings have profound
impact for clinical studies evaluating FLT3 PTK inhibitors in patients with AML and
should be considered when clinical resistance to these compounds occurs.
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8. ACKNOWLEDGMENTS
This work was carried out at the GSF- National Research Center for Environment
and Health, Munich, Germany in the K. Spiekermann group, CCG-Leukemia.
First of all I would like to thank my mother and my brother for their continuous
support in my whole life, their love and friendship. My special thank you goes to
my husband Dmitry Semikoz for his great patience and his love, without him I
wouldn’t have the possibility to make the work which I really like to.
I am very thankful to Prof. Dr. Wolfgang Hiddemann for the possibility to work in
the laboratory on my dissertation in the field “Acute myeloid leukemia”.
I am extremely grateful to Dr. Karsten Spiekermann, the best group leader, who
gave me a primary opportunity to attend research work, for his continuous support
and guidance.
I am very grateful to my colleagues Ruth Schwab, Sabine Eichenlaub, Stefanie
Geisenhof, Ralf Dirschinger, Tobias Kohl, Karin Schmieja and all other members
of the laboratory for their friendship, help, cooperation and excellent technical
assistance.
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9. PUBLICATIONS Papers: 1. Bagrintseva K., Schwab R., Kohl TM, Schnittger S., Eichenlaub S., Ellwart J.W., Hiddemann W., Spiekermann K. Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of aquired drug resistance to PTK inhibitors in FLT3-ITD transformed hematopoietic cells. Blood, 2003 Nov 6 [Epub ahead of print].
2. Spiekermann K., Bagrintseva K., Schoch C., Haferlach T., Hiddemann W., Schnittger S. (2002): A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. Blood 100, 3423-3425 3. Spiekermann K., Dirschinger R, Schwab R., Bagrintseva K., Faber F., Buske C, Schnittger S., Kelly L. M., Gilliland D. G., Hiddemann W. (2003): The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing constitutively activated FLT3. Blood 101, 1494-1504
4. Spiekermann K., Bagrintseva K., Schwab R., Schmieja K., Hiddemann W.(2003): Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells. Clin.Canc.Res. 9, 2140-2150 Abstracts: 1. Bagrintseva K., Geisenhof S, Schwab R, Eichenlaub S, Hiddemann W, Spiekermann K. [P48] Dual FLT3-ITD/TKD Mutants Found in AML confer Resistance to FLT3 PTK Inhibitors and Daunorubicin Abstract #48 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 2. Bagrintseva K, Schwab R, Schnittger S, Eichenlaub S, Hiddemann W, Spiekermann K. [P47] Acquisition of Mutations in the PTK Domain of FLT3 represents a New Molecular Mechanism of Drug Resistance to PTK Inhibitors in FLT3-ITD Transformed Cells Abstract #47 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 3. Schwab R., Bagrintseva K., Wolf U., Eichenlaub S., Hiddemann W., Spiekermann K. [P65] The Transforming Potential of FLT3-TKD Mutants Depends on Tyrosine Residues 589/591 and 597/599. Abstract #65 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 4. Bagrintseva K., Geisenhof S, Schwab R, Eichenlaub S, Hiddemann W, Spiekermann K. [2174] Dual Activating FLT3-ITD/TKD Mutations Found in patients with AML Induce Resistance to FLT3 PTK Inhibitors and Daunorubicin by Upregulation of Bcl-xL. Abstract #2174 appears in Blood, Volume 102, issue 11, November 16, 2003
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5. Bagrintseva K, Schwab R, Schnittger S, Eichenlaub S, Hiddemann W, Spiekermann K. [2175] Mutations in the Tyrosine Kinase Domain of FLT3 Define a New Molecular Mechanism of Acquired Drug Resistance to PTK Inhibitors in FLT3-ITD Transformed Cells Abstract #2175 appears in Blood, Volume 102, issue 11, November 16, 2003 6. Bagrintseva K., Schwab R., Schnittger S., Eichenlaub S., Hiddemann W., Spiekermann K. Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of aquired drug resistance to PTK inhibitors in FLT3-ITD transformed cells. DGHO (Deutsche Gesellschaft für Hämatologie und Oncologie) 2003 7.Spiekermann K., Bagrintseva K., Schoch C., Haferlach T., Hiddemann W., Schnittger S. (2002): A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. DGHO (Deutsche Gesellschaft für Hämatologie und Oncologie) 2002 8. Spiekermann K, Bagrintseva K, Schwab R, Schmieja K, Hiddemann W. [1280] Overexpression and Constitutive Activation of FLT3 Induces STAT5 Activation in Primary AML Blast Cells. #1280 Blood, Volume 101, issue 10, 2002
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10. ABBREVIATIONS AML Acute Myeloid Leukemia
MT Mutation
FLT3 fms-like tyrosine kinase-3
FCS Fetal Calf Serum
MAPK Mitogen-Activated Protein Kinase
STAT Signal Transducer and Activator of Transcription
MDS Myelodysplastic syndrom
PBS Phosphate Buffered Saline
PDGFR Platelet Derived Growth Factor Receptor
TKD Tyrosine Kinase Domain
LM Length Mutation
ITD Internal Tandem Duplication
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11. REFERENCES
Abu-Duhier, F. M., A. C. Goodeve, et al. (2001). “Identification of novel FLT-3
Asp835 mutations in adult acute myeloid leukaemia.” Br J Haematol 113(4):
983-8.
Akiyama, T., J. Ishida, et al. (1987). “Genistein, a specific inhibitor of tyrosine-
specific protein kinases.” J Biol Chem 262(12): 5592-5.
Barthe, C., M. Gharbi, et al. (2002). “Mutation in the ATP-binding site of BCR-ABL
in a patient with chronic myeloid leukaemia with increasing resistance to
STI571.” Br J Haematol 119(1): 109-11.
Blume-Jensen, P. and T. Hunter (2001). “Oncogenic kinase signalling.” Nature
411(6835): 355-65.
Branford, S., Z. Rudzki, et al. (2002). “High frequency of point mutations clustered
within the adenosine triphosphate-binding region of BCR/ABL in patients
with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia
who develop imatinib (STI571) resistance.” Blood 99(9): 3472-5.
Carow, C. E., M. Levenstein, et al. (1996). “Expression of the hematopoietic
growth factor receptor FLT3 (STK-1/Flk2) in human leukemias.” Blood
87(3): 1089-96.
Drexler, H. G. (1996). “Expression of FLT3 receptor and response to FLT3 ligand
by leukemic cells.” Leukemia 10(4): 588-99.
Druker, B. J., C. L. Sawyers, et al. (2001). “Activity of a specific inhibitor of the
BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and
acute lymphoblastic leukemia with the Philadelphia chromosome.” N Engl J
Med 344(14): 1038-42.
Druker, B. J., M. Talpaz, et al. (2001). “Efficacy and safety of a specific inhibitor of
the BCR-ABL tyrosine kinase in chronic myeloid leukemia.” N Engl J Med
344(14): 1031-7.
Fenski, R., K. Flesch, et al. (2000). “Constitutive activation of FLT3 in acute
myeloid leukaemia and its consequences for growth of 32D cells.” Br J
Haematol 108(2): 322-30.
- 80 -
Fong, T. A., L. K. Shawver, et al. (1999). “SU5416 is a potent and selective
inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that
inhibits tyrosine kinase catalysis, tumor vascularization, and growth of
multiple tumor types.” Cancer Res 59(1): 99-106.
Foran, J., A.-M. O'Farrell, et al. (2002). “An innovative single dose clinical study
shows potent inhibition of FLT3 phosphorylation by SU11248 in vivo: a
clinical and pharmacodynamic study in AML patients.” Blood 100: 559a.
Foran, J., R. Paquette, et al. (2002). “A phase I study of repeated oral dosing with
SU11248 for the treatment of patients with acute myeloid leukemia who
have failed, or are not eligible for, conventional chemotherapy.” Blood
(abstract) 100: 558a.
Gambacorti-Passerini, C., R. Barni, et al. (2000). “Role of alpha1 acid glycoprotein
in the in vivo resistance of human BCR- ABL(+) leukemic cells to the abl
inhibitor STI571.” J Natl Cancer Inst 92(20): 1641-50.
Gorre, M. E., M. Mohammed, et al. (2001). “Clinical resistance to STI-571 cancer
therapy caused by BCR-ABL gene mutation or amplification.” Science
293(5531): 876-80.
Grundler, R., C. Thiede, et al. (2003). “Sensitivity towards tyrosine kinase
inhibitors varies between different activating mutations of the FLT3
receptor.” Blood 27: 27.
Hayakawa, F., M. Towatari, et al. (2000). “Tandem-duplicated Flt3 constitutively
activates STAT5 and MAP kinase and introduces autonomous cell growth in
IL-3-dependent cell lines.” Oncogene 19(5): 624-31.
Hofmann, W. K., L. C. Jones, et al. (2002). “Ph(+) acute lymphoblastic leukemia
resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL
gene mutation.” Blood 99(5): 1860-2.
Kantarjian, H., C. Sawyers, et al. (2002). “Hematologic and cytogenetic responses
to imatinib mesylate in chronic myelogenous leukemia.” N Engl J Med
346(9): 645-52.
Karali, V., E. Dimitriadou, et al. (2002). “ITD and Asp835 mutation of FLT-3 gene
can occur simultaneously in AML patients.” Blood 100(11): 197b.
Kelly, L. M., Q. Liu, et al. (2002). “FLT3 internal tandem duplication mutations
associated with human acute myeloid leukemias induce myeloproliferative
disease in a murine bone marrow transplant model.” Blood 99(1): 310-8.
- 81 -
Kelly, L. M., J. C. Yu, et al. (2002). “CT53518, a novel selective FLT3 antagonist
for the treatment of acute myelogenous leukemia (AML).” Cancer Cell 1(5):
421-32.
Kiyoi, H., T. Naoe, et al. (1999). “Prognostic implication of FLT3 and N-RAS gene
mutations in acute myeloid leukemia.” Blood 93(9): 3074-80.
Kiyoi, H., T. Naoe, et al. (1997). “Internal tandem duplication of FLT3 associated
with leukocytosis in acute promyelocytic leukemia. Leukemia Study Group
of the Ministry of Health and Welfare (Kohseisho).” Leukemia 11(9): 1447-
52.
Kiyoi, H., M. Towatari, et al. (1998). “Internal tandem duplication of the FLT3 gene
is a novel modality of elongation mutation which causes constitutive
activation of the product.” Leukemia 12(9): 1333-7.
Kottaridis, P. D., R. E. Gale, et al. (2001). “The presence of a FLT3 internal
tandem duplication in patients with acute myeloid leukemia (AML) adds
important prognostic information to cytogenetic risk group and response to
the first cycle of chemotherapy: analysis of 854 patients from the United
Kingdom Medical Research Council AML 10 and 12 trials.” Blood 98(6):
1752-9.
le Coutre, P., E. Tassi, et al. (2000). “Induction of resistance to the Abelson
inhibitor STI571 in human leukemic cells through gene amplification.” Blood
95(5): 1758-66.
Levis, M., K. F. Tse, et al. (2001). “A FLT3 tyrosine kinase inhibitor is selectively
cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem
duplication mutations.” Blood 98(3): 885-7.
Longley, B. J., M. J. Reguera, et al. (2001). “Classes of c-KIT activating mutations:
proposed mechanisms of action and implications for disease classification
and therapy.” Leuk Res 25(7): 571-6.
Lyman, S. D. and S. E. Jacobsen (1998). “c-kit ligand and Flt3 ligand:
stem/progenitor cell factors with overlapping yet distinct activities.” Blood
91(4): 1101-34.
Ma, Y., S. Zeng, et al. (2002). “The c-KIT mutation causing human mastocytosis is
resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic
site mutations show different inhibitor sensitivity profiles than wild-type
kinases and those with regulatory-type mutations.” Blood 99(5): 1741-4.
- 82 -
Mahon, F. X., M. W. Deininger, et al. (2000). “Selection and characterization of
BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase
inhibitor STI571: diverse mechanisms of resistance.” Blood 96(3): 1070-9.
Matthews, W., C. T. Jordan, et al. (1991). “A receptor tyrosine kinase cDNA
isolated from a population of enriched primitive hematopoietic cells and
exhibiting close genetic linkage to c-kit.” Proc Natl Acad Sci U S A 88(20):
9026-30.
Mizuki, M., R. Fenski, et al. (2000). “Flt3 mutations from patients with acute
myeloid leukemia induce transformation of 32D cells mediated by the Ras
and STAT5 pathways.” Blood 96(12): 3907-14.
Moreno, I., G. Martin, et al. (2003). “Incidence and prognostic value of FLT3
internal tandem duplication and D835 mutations in acute myeloid leukemia.”
Haematologica 88(1): 19-24.
Nakao, M., S. Yokota, et al. (1996). “Internal tandem duplication of the flt3 gene
found in acute myeloid leukemia.” Leukemia 10(12): 1911-8.
Reilly, J. T. (2002). “Class III receptor tyrosine kinases: role in leukaemogenesis.”
Br J Haematol 116(4): 744-57.
Rombouts, W. J., I. Blokland, et al. (2000). “Biological characteristics and
prognosis of adult acute myeloid leukemia with internal tandem duplications
in the Flt3 gene.” Leukemia 14(4): 675-83.
Rosnet, O., H. J. Buhring, et al. (1996). “Human FLT3/FLK2 receptor tyrosine
kinase is expressed at the surface of normal and malignant hematopoietic
cells.” Leukemia 10(2): 238-48.
Rosnet, O., S. Marchetto, et al. (1991). “Murine Flt3, a gene encoding a novel
tyrosine kinase receptor of the PDGFR/CSF1R family.” Oncogene 6(9):
1641-50.
Roumiantsev, S., N. P. Shah, et al. (2002). “Clinical resistance to the kinase
inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the
Abl kinase domain P-loop.” Proc Natl Acad Sci U S A 99(16): 10700-5.
Savage, D. G. and K. H. Antman (2002). “Imatinib mesylate--a new oral targeted
therapy.” N Engl J Med 346(9): 683-93.
Sawyers, C. L. (2001). “Research on resistance to cancer drug Gleevec.” Science
294(5548): 1834.
- 83 -
Schnittger, S., I. Boell, et al. (2002). “FLT3D835/I836 Point Mutations in Acute
Myeloid Leukemia: Correlation to Cytogenetics, Cytomorphology, and
Prognosis in 1229 Patients.” Blood 100: 329a.
Schnittger, S., U. Kinkelin, et al. (2000). “Screening for MLL tandem duplication in
387 unselected patients with AML identify a prognostically unfavorable
subset of AML.” Leukemia 14(5): 796-804.
Schnittger, S., C. Schoch, et al. (2002). “Analysis of FLT3 length mutations in 1003
patients with acute myeloid leukemia (AML): Correlation to cytogenetics,
FAB subtype, and prognosis in the AMLCG study, and usefulness as a
marker for detection of minimal residual disease.” Blood 100: 59-66.
Schnittger, S., C. Schoch, et al. (2001). “Rapid, Simple and Reliable Detection of
FLT3 Asp835 Point Mutations in Acute Myeloid Leukemia and Identification
of a New Length Mutation In Exon 20.” Blood (abstract) 98: 107a.
Schnittger, S., C. Schoch, et al. (2001). “FLT3-LM and MLL-PTD as Markers for
PCR-Based Detection of Minimal Residual Disease (MRD) in AML with
Normal Karyotype.” Blood 98: 581a.
Shah, N. P., J. M. Nicoll, et al. (2002). “Multiple BCR-ABL kinase domain
mutations confer polyclonal resistance to the tyrosine kinase inhibitor
imatinib (STI571) in chronic phase and blast crisis chronic myeloid
leukemia.” Cancer Cell 2(2): 117-25.
Smith, B. D., M. Levis, et al. (2002). “Single agent CEP-701, a novel FLT-3
inhibitor, shows initial response in patients with refractory acute myeloid
leukemia.” Blood (abstract) 100: 85a.
Spiekermann, K., R. J. Dirschinger, et al. (2003). “The protein tyrosine kinase
inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in
AML-derived cell lines expressing a constitutively activated FLT3.” Blood
101(4): 1494-504.
Spiekermann, K., F. Faber, et al. (2002). “The protein tyrosine kinase inhibitor
SU5614 inhibits VEGF-induced endothelial cell sprouting and induces
growth arrest and apoptosis by inhibition of c-kit in AML cells.” Exp Hematol
30(7): 767-73.
Stone, R., V. Klimek, et al. (2002). “PKC412, an oral FLT3 inhibitor, has activity in
mutant FLT3 acute myeloid leukemia (AML): a phase II clinical trial.” Blood
(abstract) 100: 86a.
- 84 -
Thiede, C., C. Steudel, et al. (2001). “Analysis of FLT3-activating mutations in 712
patients with acute myelogenous leukemia: High incidence in FAB-subtype
M5 and identification of subgroups with poor prognosis.” Ann Hematol.
Thiede, C., C. Steudel, et al. (2002). “Analysis of FLT3-activating mutations in 979
patients with acute myelogenous leukemia: association with FAB subtypes
and identification of subgroups with poor prognosis.” Blood 99(12): 4326-35.
Tse, K. F., G. Mukherjee, et al. (2000). “Constitutive activation of FLT3 stimulates
multiple intracellular signal transducers and results in transformation.”
Leukemia 14(10): 1766-76.
Tse, K. F., E. Novelli, et al. (2001). “Inhibition of FLT3-mediated transformation by
use of a tyrosine kinase inhibitor.” Leukemia 15(7): 1001-10.
van Oosterom, A. T., I. Judson, et al. (2001). “Safety and efficacy of imatinib
(STI571) in metastatic gastrointestinal stromal tumours: a phase I study.”
Lancet 358(9291): 1421-3.
von Bubnoff, N., F. Schneller, et al. (2002). “BCR-ABL gene mutations in relation
to clinical resistance of Philadelphia-chromosome-positive leukaemia to
STI571: a prospective study.” Lancet 359(9305): 487-91.
Weisberg, E., C. Boulton, et al. (2002). “Inhibition of mutant FLT3 receptors in
leukemia cells by the small molecule tyrosine kinase inhibitor PKC412.”
Cancer Cell 1(5): 433-43.
Weisberg, E. and J. Griffin (2000). “Mechanism of resistance to the ABL tyrosine
kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines.”
Blood 95(11): 3498-505.
Whitman, S. P., K. J. Archer, et al. (2001). “Absence of the Wild-Type Allele
Predicts Poor Prognosis in Adult de Novo Acute Myeloid Leukemia with
Normal Cytogenetics and the Internal Tandem Duplication of FLT3: A
Cancer and Leukemia Group B Study.” Cancer Res 61(19): 7233-9.
Wybenga-Groot, L. E., B. Baskin, et al. (2001). “Structural basis for autoinhibition
of the ephb2 receptor tyrosine kinase by the unphosphorylated
juxtamembrane region.” Cell 106(6): 745-57.
Xu, F., T. Taki, et al. (1999). “Tandem duplication of the FLT3 gene is found in
acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not
in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in
children.” Br J Haematol 105(1): 155-62.
- 85 -
Yamamoto, Y., H. Kiyoi, et al. (2001). “Activating mutation of D835 within the
activation loop of FLT3 in human hematologic malignancies.” Blood 97(8):
2434-2439.
Yee, K., A. O'Farrell, et al. (2002). “SU5416 and SU5614 inhibit kinase activity of
wild-type and mutant FLT3 receptor tyrosine kinase.” Blood 100(8): 2941-9.
Zermati, Y., P. De Sepulveda, et al. (2003). “Effect of tyrosine kinase inhibitor
STI571 on the kinase activity of wild-type and various mutated c-kit
receptors found in mast cell neoplasms.” Oncogene 22(5): 660-4.
- 86 -
12. CURRICULUM VITAE
KSENIA BAGRINTSEVA
Date of birth: 25.07.1975 Adress: Joh-Seb-Bach str. 9, 80637 Place of birth: Moscow, Russia Munich, Germany Marital status: married Phone: (+49) 175 6360017 Citizenship: Russian E-mail [email protected]
Education 1/04- present Postdoctoral position GSF (National Research Center for Environment and Health), Clinical Cooperative Group ‘‘Leukemia’’, Ludwig-Maximilian University Munich, Germany 7/00- 12/03 MD, graduate student GSF (National Research Center for Environment and Health), Clinical Cooperative Group ‘‘Leukemia’’, Ludwig-Maximilian University Munich, Germany Graduate Student; Supervisor: Prof. Dr. W. Hiddemann Dissertation: The role of the FLT3 mutations in AML. The possible mechanisms of the drug resistance development to specific PTK inhibitors. Found and characterized a new mutations in FLT3:840GS and V592A. Generated PTK inhibitor resistant cell lines and characterized the mechanisms of these resistance. 9/93-7/99 Doctor of Medicine Medical Faculty, Russian State Medical University Moscow, Russia 9/91-7/93 Diploma Nurse with honor Central clinical hospital of the Government of Russian Federation Moscow, Russia
Experimental Skills DNA/RNA: recombinant DNA techniques, plasmid DNA isolation, PCR, mutagenesis; Protein: immunoprecipitation analysis, Western blot analysis; Cell culture: basic cell culture techniques, transient transfection techniques, retroviral infection, basic FACS analysis, apoptose assay. Language Skills
Russian (native), English (fluent), German (fluent; Diploma: “Kleines Deutsches Sprachdiplom“, Goethe-Institut, Munich, Germany) Computer Skills Microsoft Word, Excel, Power Point, Sigma Plot, Adobe Illustrator, Adobe Photoshop, Winmdi, BioEdit, EndNote.
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Activities
2/98-2/99 Russian Red Cross medical help at home to elderly patients in Moscow, Russia 9/98-6/99 Diploma in Therapeutic Massage ; Moscow, Russia 9/85-5/91 School of Music: class piano (Diploma); Moscow, Russia Participation in scientific meetings:
Posters: 1. Bagrintseva K., Geisenhof S, Schwab R, Eichenlaub S, Hiddemann W, Spiekermann K. [P48] Dual FLT3-ITD/TKD Mutants Found in AML confer Resistance to FLT3 PTK Inhibitors and Daunorubicin Abstract #48 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 2. Bagrintseva K, Schwab R, Schnittger S, Eichenlaub S, Hiddemann W, Spiekermann K. [P47] Acquisition of Mutations in the PTK Domain of FLT3 represents a New Molecular Mechanism of Drug Resistance to PTK Inhibitors in FLT3-ITD Transformed Cells Abstract #47 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 3. Schwab R., Bagrintseva K., Wolf U., Eichenlaub S., Hiddemann W., Spiekermann K. [P65] The Transforming Potential of FLT3-TKD Mutants Depends on Tyrosine Residues 589/591 and 597/599. Abstract #65 appears in Annals of Hematology, Suppl1 to Vol 83 (2004) 4. Bagrintseva K., Geisenhof S, Schwab R, Eichenlaub S, Hiddemann W, Spiekermann K. [2174] Dual Activating FLT3-ITD/TKD Mutations Found in patients with AML Induce Resistance to FLT3 PTK Inhibitors and Daunorubicin by Upregulation of Bcl-xL. Abstract #2174 appears in Blood, Volume 102, issue 11, November 16, 2003 5. Bagrintseva K, Schwab R, Schnittger S, Eichenlaub S, Hiddemann W, Spiekermann K. [2175] Mutations in the Tyrosine Kinase Domain of FLT3 Define a New Molecular Mechanism of Acquired Drug Resistance to PTK Inhibitors in FLT3-ITD Transformed Cells Abstract #2175 appears in Blood, Volume 102, issue 11, November 16, 2003 6. Bagrintseva K., Schwab R., Schnittger S., Eichenlaub S., Hiddemann W., Spiekermann K. Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of aquired drug resistance to PTK inhibitors in FLT3-ITD transformed cells. DGHO (Deutsche Gesellschaft für Hämatologie und Oncologie)2003 7.Spiekermann K., Bagrintseva K., Schoch C., Haferlach T., Hiddemann W., Schnittger S. (2002): A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. DGHO (Deutsche Gesellschaft für Hämatologie und Oncologie)2002 8. Spiekermann K, Bagrintseva K, Schwab R, Schmieja K, Hiddemann W. [1280] Overexpression and Constitutive Activation of FLT3 Induces STAT5 Activation in Primary AML Blast Cells. #1280 Blood, Volume 101, issue 10, 2002 Grant 5/02-11/02 personal grant from Jose-Carreras Foundation; Munich, Germany.
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Publications 1. Bagrintseva K., Schwab R., Kohl TM, Schnittger S., Eichenlaub S., Ellwart J.W., Hiddemann W., Spiekermann K. Mutations in the tyrosine kinase domain of FLT3 define a new molecular mechanism of aquired drug resistance to PTK inhibitors in FLT3-ITD transformed hematopoietic cells. Blood, 2003 Nov 6 [in press]. 2. Spiekermann K., Bagrintseva K., Schoch C., Haferlach T., Hiddemann W., Schnittger S. (2002): A new and recurrent activating length mutation in exon 20 of the FLT3 gene in acute myeloid leukemia. Blood 100,3423-3425 3. Spiekermann K., Dirschinger R, Schwab R., Bagrintseva K., Faber F., Buske C, Schnittger S., Kelly L. M., Gilliland D. G., Hiddemann W. (2003): The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing constitutively activated FLT3. Blood 101, 1494-1504 4. Spiekermann K., Bagrintseva K., Schwab R., Schmieja K., Hiddemann W.(2003): Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells. Clin.Canc.Res. 9, 2140-2150
References
1. Prof. Dr. med. Wolfgang Hiddemann, Head of Department of Medicine III, University Hospital Grosshadern, Ludwig-Maximilians University, Clinical Cooperative Group “Leukemia”; GSF-National Research Center for Environment and Health Marchioninistr. 15, 81377 Munich, Germany. Tel. +49(89)7095 2551, e-mail: [email protected]
2. Dr. med. Karsten Spiekermann, GSF (National Research Center for Environment and Health), Clinical Cooperative Group ‘‘Leukemia’’, Marchioninistr. 25, Munich, Germany. Tel. +49(89) 7099423, e-mail: [email protected]
3. Prof. Dr. med. Stefan K. Bohlander, GSF (National Research Center for Environment and Health), Clinical Cooperative Group ‘‘Leukemia’’, Marchioninistr. 25, Munich, Germany. Tel. +49(89) 7099357, e-mail: [email protected]